@Copyright 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3401-0045 $02.00
ATP-Dependent Strontium Uptake by Basolateral Membrane Vesicles from Rat Renal Cortex in the Absence or Presence of Calcium NAOKO SUGIHIRA,* YASUNOBU AOKI, AND KAZUO T. SUZUKI
National Institute for Environmental Studies, Tsukuba, /barak/305, Japan Received May 28, 1991; Accepted July 21, 1991
ABSTRACT ATP-dependent Sr 2+ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent S r 2 + uptake and C a 2 + uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for C a 2+ and S r 2 + uptake. The apparent Km and V,,~x of ATP-dependent Sr 2 + uptake were both higher than those for C a 2 + uptake. ATP-dependent S r 2 + uptake by BLM vesicles diminished in the presence of 0.1 I~M Ca 2+ and was more markedly inhibited by 1 laM C a 2 + . Hill plots of S r 2+ uptake data with and without 0.1 ~M C a 2 + showed that the cooperative behavior of S r 2 + uptake was not changed by C a 2 +. In the presence of 0.1 BM C a 2 +, the affinity of the transport system for Sr2+ and the velocity of S r 2 + uptake in the BLM were both decreased. However, the rate of C a 2+ uptake was not diminished by S r 2 + concentrations of < 1.6 p~M. These results suggest that C a 2 § is preferentially transported in the renal cortex BLM w h e n C a 2 + and S r 2 + are present at the same time. Index Entries Strontium; calcium; ATP-dependent uptake of cation; rat renal cortex; basolateral membrane vesicles; renal discrimination. *Author to w h o m all c o r r e s p o n d e n c e a n d reprint requests should b e addressed.
Biological Trace Element Research
45
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
46
E G T A , ethyleneglycol-bis-(f3-aminoethyl acid; HEDTA, N-hydroxyethylenediaminetetraacetic acid; (Mg2+/Ca2+)-ATPase, Mg2+-dependent Ca 2+ -stimulated adenosine triphosphatase; Mops, 4-morpholinepropanesulfonic acid; (Na +/K * )-ATPase, (Na +/K +)-stimulated adenosine triphosphatase. Abbreviations
ether)-N,N,N',N'-tetraacetic
INTRODOCTION The chemical properties of strontium (Sr) are very similar to those of calcium (Ca), and the metabolism and distribution of Sr in the body closely resemble those of Ca (1). However, discrimination of Sr in favor of Ca has been shown to occur during processes in which the animal body absorbs these elements from the environment and utilizes them (2-4). We have investigated renal discrimination between Sr and Ca during reabsorption in both experimental and diagnostic studies (5-8). Our previous observations suggested that renal tubules in rats (5-7) and humans (8) discriminate against Sr in favor of Ca by strictly regulating the reabsorption rate of Sr compared to that of Ca. However, the mechanisms by which the renal tubular cells discriminate between these two elements are not known. Although Ca reabsorption by the renal tubules is one of the major regulatory mechanisms for maintaining Ca homeostasis, its mechanisms have not been fully elucidated. It is generally assumed that Ca 2+ enters the tubular cells across the brush-border membrane from the tubular lumen by diffusion, because renal cytosolic free Ca 2§ concentrations are in the submicromolar range (9) and the membrane potential in the interior of the cell is negative. In contrast, C a 2 + has to be actively transported out of the cell across the basolateral segment of the plasma membrane, since the Ca 2+ electrochemical gradient at this membrane is reversed. Two different transport systems for C a 2 + in the basolateral membrane (BLM) have been proposed: One is a Na + / C a 2 + exchange process (10-12), and the other is ATP-dependent C a 2+ transport (11,13,14). ATP-dependent Ca 2+ transport has been shown to be mediated by a Ca2+-stimulated Mg2+-dependent ATPase in isolated BLM from rat renal cortex (13,14). van Heeswijk et al. (14) proposed that the (Mg2+/ Caa+)-ATPase system in rat renal cortex cells plays a primary role in Ca 2+ efflux. Snowdowne and Borle (15) reported that the Na +/Ca 2+ exchange is much less active than the ATPase system. There is a possibility that the active transport systems for Ca 2+ in renal BLM, described above, may be involved in the discrimination mechanisms between Sr and Ca in the renal tubules. In the present study, we examined ATP-dependent S r 2+ transport in BLM isolated from the renal cortex in the absence and presence of C a 2 + in order to find information that might help clarify the discrimination mechanisms between the two elements. Biological Trace Element Research
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Renal Basolateral Membrane Sr2+ Transport
47
MATERIALS A N D METHODS Preparation of Renal Bl..a4 Vesicles
The renal cortices from six male Wistar rats (200-250 g) were used for each preparation. Basolateral membrane vesicles were isolated by a Percoll gradient procedure according to the method of Gmaj et al. (16). The isolated BLM were suspended in about I mL of a storage solution, which contained 100 mM mannitol, 100 mM KC1, 20 mM K'Mops (pH 7.0), 3 mM MgC12, 5 mM EGTA, I mM dithiothreitol, 1 mM ATP, and 15% (w/v) glycerol. The suspension was divided into 0.2-mL samples and stored in a liquid-nitrogen tank. Before each experiment, the membranes were thawed on ice, diluted to 1.5 mL with 100 mM KC1, 50 mM K-Mops (pH 7.4), 5 mM MgCI2, and 1 mM dithiothreitol, and centrifuged for 15 min. The washing step was repeated once, and the final membrane pellet was suspended in 0.3 mL of the same solution. Neither the ATP-dependent transport activity of Ca 2+ nor of Sr 2+ was changed by the freezing, thawing, and washing procedures. The activity of the ouabain-sensitive (Na +/K +)-ATPase, an enzyme marker for the BLM, was determined according to the method of Mircheff and Wright (17). Protein concentration was measured by the method of Lowry et al. (18) using bovine serum albumin as a standard. Ca 2 § or Sr 2 § Uptake Measurements
ATP-dependent uptake of Ca 2 + and Sr 2 + was determined according to the Ca 2+ uptake measurement method reported by Gmaj et al. (16). The kinetics of Ca 2+ and Sr 2+ transport was obtained from measurements of the initial rate of uptake for each ion (16). The incubation medium contained 100 mM KC1, 50 mM K-Mops (pH 7.4), 0.2 mM HEDTA, 0.5 mM EGTA, 10 b~M oligomycin, a calculated amount of MgC12 to keep the free Mg 2+ concentrations at about 4.7 mM, and calculated amounts of CaC12 or SrC12, or both. Finally, the medium contained 15 ~Ci 45Ca/mL and/or 2 p~Ci 85Sr/mL. The total amount of metal that had to be added to this system with several ligands and metal ions in order to reach the desired free metal ion concentration was calculated using the equation described by Bulos and Sacktor (19). The BLM suspension (20 b~L)was added to 100 b~L of the incubation medium, and the mixture was preincubated for 4 min at 37~ The uptake reaction was started by the addition of 5 ILL of 100 mM Mg-ATP to the medium. At given time intervals, 20-p,L samples were withdrawn and filtered through cellulose nitrate filters (0.45-p,m pore diameter). The filters were washed twice with 4-mL portions of ice-cold stopping solution containing 150 mM KC1, 20 mM K.Mops (pH 7.4), and 2 mM EGTA. The amount of radioactive 45Ca retained on the filters was measured in a liquid scintillation counter, and radioactive 85Sr was measured in a 3~ scintillation counter. The initial rates (v0) of Ca 2§ (or Sr 2+) uptake Biological Trace Element Research
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
48 =
6
e~
a.S
2
~
0
"
,
I
0.2
0.0
,
I
0.4
,
I
0.6
,
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0.8
,
I
1.0
,
1.2
Free Ca 2, concentration (~tM) Fig. 1. Relationship between free C a 2+ concentration and the ATP-dependent C a 2+ uptake rate in basolateral membrane vesicles from rat kidney cortex. Each data point represents the mean + SE for three to seven observations in a single representative experiment. were calculated as the slopes of the regression lines of Ca 2+ (or Sr 2+) u p t a k e vs time.
RESULTS The specific activity of the ouabain-sensitive (Na +/K +)-ATPase, the e n z y m e m a r k e r for the BLM, in the BLM preparations was 1.37 + 0.13 p,mol Pi/min'mg protein (mean + SE, n = 3), indicating a 26-29-fold e n r i c h m e n t relative to the renal cortex h o m o g e n a t e . Ca 2 + u p t a k e from a m e d i u m containing 50 I~M CaCI2 a n d Sr 2 + u p t a k e from a m e d i u m containing 50 laM SrC12 w e r e both m e a s u r e d in BLM vesicles. In the presence of ATP, the BLM vesicles were f o u n d to accumulate Ca 2 + a n d Sr 2 § to levels exceeding those observed in the absence of ATP after 10 m i n of incubation by fourfold a n d eightfold, respectively. This indicates that A T P - d e p e n d e n t transport of S r 2 + as well as Ca 2 § occurs in the renal BLM. The kinetics of A T P - d e p e n d e n t Ca 2 + a n d Sr 2 + transport in the BLM vesicles is s h o w n in Figs. 1 a n d 2, respectively. Both Ca 2+ a n d Sr 2+ u p t a k e rates were increased d e p e n d i n g on cation concentration a n d were saturated at concentrations of cations h i g h e r than 0.35 a n d 1.25 t~M, respectively. The addition of 10 I~M vanadate, an inhibitor of (Mg 2+ / Ca~+)-ATPases, into the incubation m e d i u m r e d u c e d A T P - d e p e n d e n t Ca 2 + u p t a k e rates to 44-70% of those observed in the absence of vanadate. In the presence of 10 t~M vanadate, A T P - d e p e n d e n t Sr 2 + u p t a k e rates also d i m i n i s h e d to < 10% of those seen in the absence of vanadate. The relationship b e t w e e n free Sr 2+ concentration a n d ATPBiological Trace Element Research
VoL 34, 1992
Renal Basolateral Membrane Sr 2 + Transport
49
3 ~
8
O 6
~4
,
I
~
I
1.0
U.O
,
I
2.0
3.0
Free Sr 2§ concentration (~M) Fig. 2. Relationship between free Sr 2+ concentration and the ATP-dependent Sr 2 + uptake rate in basolateral membrane vesicles from rat kidney cortex. Each data point represents the mean _+ SE for three to six observations in a single representative experiment.
,~
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Free Sr 2+ concentration (~tM) Fig. 3. Relationship between free Sr 2§ concentration and the ATP-dependent Sr 2+ uptake rate in basolateral membrane vesicles from rat kidney cortex in the absence of Ca 2 + (C)) or in the presence of 0.1 pLM (m) or 1 p.M (&) free Ca 2+. Each data point represents the mean + SE for three to seven observations in a single representative experiment. d e p e n d e n t Sr 2 + u p t a k e in the presence of 0.1 ~M or 1 ~M free Ca 2 + is s h o w n in Fig. 3. The free Ca 2+ concentration in the cytosol of the proximal tubular e p i t h e l i u m is k n o w n to be approx 0.1 ~M (9). U n d e r this experimental condition, the ratios (w/w) of total Sr concentration to total Ca concentration in the reaction mixture were 0.01-1.63, w h i c h were about 10-100 times those observed in the b o d y (20). The presence of Biological Trace Element Research
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
50
%
h...I
.1Ill! I
-2
-2
-1
i
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i
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Log [M 2+] Fig. 4. Hill plots of the kinetic data shown in Figs. 1-3. 0 - - 0 , ATPdependent C a 2+ uptake; 9169 ATPdependent Sr 2 + uptake in the absence of Ca 2 +; " - - ' , ATP-dependent Sr 2 + uptake in the presence of 0.1 ~M C a R+. [M 2+] represents the free cation concentration. The V,,,~xvalue used for these calculations was derived from the Lineweaver-Burk plot in the high ranges of cation concentration. 0.1 ~M Ca 2+ inhibited ATP-dependent Sr 2+ uptake compared with uptake observed in the absence of C a 2 + . The addition of 1 ~M C a 2 + further inhibited ATP-dependent Sr 2 + uptake. The data s h o w n in Figs. 1-3 were transformed to Hill plots (Fig. 4), since the Lineweaver-Burk reciprocal plots of the data were not completely linear (not shown). Sr 2+ uptake data in the presence of I ~M Ca 2+ were not transformed to a Hill plot, because the Sr ~+ uptake rate was not saturated in the concentration ranges examined (Fig. 3). The Vmaxvalues used for the calculations in the Hill plots were derived from the respective Lineweaver-Burk plots in the high-cation concentration range. All the Hill plots yielded straight lines (Ca 2+ uptake data, r = 0.80; Sr 2+ uptake data in the absence of Ca 2+, r = 0.94; Sr 2+ uptake data in the presence of 0.1 ~M Ca 2 +, r = 0.98). The slopes (Hill coefficients, nHil0 of the three lines were similar, in the range of 1.1-1.2. The kinetic parameters of ATP-dependent Ca 2 + uptake, and those of Sr 2 + uptake in the absence and presence of 0.1 ~M Ca 2 + are summarized in Table 1. The apparent Km (for the free metal ion) and Vmaxvalues of ATP-dependent Sr 2 + uptake in the absence of Ca 2 + were higher than those for C a 2+ uptake. The Km value for S r 2+ during ATP-dependent
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Renal Basolateral Membrane Sr 2 + Transport
51
Table 1 Sr 2+ uptake ~'
Km (p,M) Wm a x (nmol/min 9 nni,
protein)
Ca 2+ uptake
- C a 2+
+ C a 2+
0.0548 +_ 0.0157
0.871 _ 0.344
1.99 _ 0.28
4.66 +_ 0.39 1.16 +_ 0.07
12.9 + 4.0 1.23 + 0.07
7.02 + 0.30 1.10 _+ 0.02
Table 2
S r 2+
C a 2+ uptake rate, nmol/min 9 mg protein
concentration, p.M 0 0.4 0.8 1.6 3.2 6.4 10.0
2.60 1.78 2.50 1.66 1.26 0.756 0.695
+ 0.17 _+ 0.05 +_ 0.12 + 0.20 b _+ 0.26 ~' _+ 0.062 ~' +_ 0.01if'
Table 3
Sr 2+ concentration, ~M 0 0.8 1.6 3.2 6.4 10.0 20.0
C a 2+ uptake rate, nmol/min 9 protein
3.16 3.54 3.26 3.28 2.59 2.92 2.12
+ 0.44 + 0.16 + 0.19 +- 0.31 _+ 0.05 _+ 0.23 +_ 0.25
Sr 2+ u p t a k e increased, a n d the V,~ x d e c r e a s e d w i t h the a d d i t i o n of 0.1 p,M Ca 2 +. The effects of Sr 2 + o n A T P - d e p e n d e n t Ca 2 + u p t a k e b y BLM vesicles in the p r e s e n c e of 0.1 p,M Ca 2+ w e r e also e x a m i n e d (Table 2). The Ca 2 + u p t a k e rate w a s n o t affected b y Sr 2 + at c o n c e n t r a t i o n s l o w e r t h a n 1.6 laM, b u t it w a s i n h i b i t e d b y Sr at c o n c e n t r a t i o n s h i g h e r t h a n a n d equal to 1.6 ~M. The effects of Sr 2 + o n A T P - d e p e n d e n t Ca z§ u p t a k e in t h e p r e s e n c e of I ~ M free Ca 2+ are s h o w n in Table 3. T h e ATPd e p e n d e n t Ca 2 + u p t a k e rate w a s n o t c h a n g e d by Sr 2+ at a n y of the c o n c e n t r a t i o n s t e s t e d (-< 20 ~M).
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Sugihira, Aoki, and Suzuki
DISCUSSION The basolateral segment of the renal tubular plasma membrane possesses an active transport system for Ca 2+ (10,11,13,14,21). This mechanism is thought to regulate the intracellular Ca 2+ concentration and to be involved in Ca reabsorption by the renal tubules (10). BLM vesicles isolated from rat renal cortex have also been reported to have ATP-dependent uptake of Ca 2+ (11,13,14). However, no mechanism of Sr reabsorption by the renal tubule or of discrimination between Sr and Ca during the reabsorption step had been elucidated. Strontium, unlike other divalent cations, has been shown to share a number of active membrane transport systems (22). For example, Sr 2+ has been shown to accumulate in vesicles derived from the sarcoplasmic reticulum of skeletal muscle at the expense of ATP hydrolysis as well as Ca 2§ uptake (23). Human red cells also have an ATP-dependent ion pump that transports not only Ca 2+, but also Sr 2. (24). The operation of the pump is also connected to the activity of a transport ATPase (22). In the present study, ATP-dependent uptake of Sr ~+, as well as that of Ca 2+, was observed in BLM vesicles derived from the renal cortex. This Sr 2+ uptake system is highly sensitive to vanadate. Vanadate is known to inhibit ATP-dependent Ca 2+ transport and (Mg2+/Ca 2+) ATPase activities in BLM from rat renal cortex (13,25)9 These findings suggest the possibility that Sr 2+ was taken up into the BLM vesicles by the same mechanisms as Ca 2§ In the presence of 0.1 ~M Ca 2+, the ATP-dependent Sr 2+ uptake rate was inhibited (Fig. 3). Hill plots of these data indicate similarities in the kinetic behavior of Ca 2 + and Sr 2 + uptake, and in Sr 2+ uptake in the presence and absence of 0.1 ~M Ca 2 + (Fig. 4). These results suggest that the cooperative behaviors of ATP-dependent C a 2 + and S r 2 + transport in BLM are similar and that the inhibition of ATP-dependent Sr 2+ transport by 0.1 ~M C a 2+ is not an allosteric effect. Kinetic parameters obtained for ATP-dependent Ca 2+ uptake by BLM vesicles were in accordance with those previously published (14,25). The K,, of ATP-dependent S r 2+ uptake was higher than that of the Ca 2+ uptake (Table 1), indicating the lower affinity of the BLM transport system for S r 2 +. These observations in the BLM vesicles from rat renal cortex are in agreement with those reported for an ATPase in the sarcoplasmic reticulum, the affinity of which was 12-14 times higher for Ca 2+ than for Sr 2§ (26). The V,,a~ for ATPase activities in skeletal sarcoplasmic reticulum vesicles was shown to be higher in the presence of Sr z + than in the presence of Ca 2+ (26). The author suggested that this could be explained by a lower affinity of the internal binding sites on the ATPase for Sr 2+ as compared to their affinity for C a 2+ and that this decreases feedback inhibition of the ATPase in sarcoplasmic reticulum vesicles (23). The higher Vmaxof ATP-dependent S r 2 + uptake compared
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Renal Basolateral Membrane Sr 2 . Transport
53
to that of Ca 2 + uptake that we obtained (Table 1) might also be explained by this mechanism. In erythrocyte ghosts, Ca 2 + is transported almost exclusively w h e n C a 2 + and Sr 2 + are present at the same time (24). In our results, ATPd e p e n d e n t S r 2 + uptake in BLM vesicles from the renal cortex was diminished by 0.1 ~M Ca 2§ and was even more markedly inhibited by 1 ~M Ca 2 + (Fig. 3). In the presence of 0.1 I~M Ca 2 +, the apparent Km of Sr 2 + uptake increased compared with that observed in the absence of Ca 2 § (Table 1). In addition to this, the Vmax of Sr 2 + uptake was diminished by the presence of 0.1 ~M Ca 2 +, which may be owing to feedback inhibition of the ATPase by Ca 2 4, possibly by the mechanism described above. We observed that 0.1 ~M free Ca 2 + , which is identical to the estimated cytosolic free Ca 2+ concentration in kidney cells, decreases the affinity of Sr 2 § for the transport system and the velocity of Sr 2 + uptake in the BLM. In the experiment in which the incubation m e d i u m contained 0.1 ~M Ca 2+ and various concentrations of free Sr 2+, the ATP-dependent Ca 2+ uptake rate was not changed by Sr 2+ concentrations of ~ 1.6 ~M (Table 2). In the presence of a higher concentration of Ca 2+ (1 ~M), the ATP-dependent Ca 2 § uptake rate was not decreased by Sr 2 § at any of the concentrations examined (Table 3). Our results suggest that Ca 2+ is preferentially transported in the BLM from rat renal cortex w h e n Ca 2 + and Sr 2 + are present at the same time, even though the ratios (w/w) of total Sr concentration to total Ca concentration in the reaction mixture are 10-100 times those observed in the body. It is likely that this preferential transport of Ca 2 + in BLM may contribute to the discrimination mechanisms between Sr and Ca during the reabsorption process along the proximal tubule.
ACKNOWLEDGMENT We wish to thank Dr. M. Murakami for his encouragement.
REFERENCES 1. R. H. Wasserman, E. M. Romney, M. W. Skougstad, and R. Siever, in Geochemistry and the Environment. vol. 2, National Academy of Science, Washington, D.C., 1977, pp. 73--87. 2. G. W. Dolphin and I. S. Eve, Phys. Med. Biol. 8, 193-203 (1963). 3. C. L. Comar and R. H. Wasserman, in Mineral Metabolism: An Advanced Treatise, C. L. Comar and F. Bronner, eds., Academic, New York, 1964, pp. 523-572. 4. C. L. Comar, in Strontium Metabolism, J. M. A. Lenihan, J. F. Loutit, and J. H. Martin, eds., Academic, New York, 1967, pp. 17-31. 5. N. Sugihira and K. T. Suzuki, Biol. Trace Elem. Res. 22, 71-82 (1989). 6. N. Sugihira and K. T. Suzuki, Trace Elem. Med. 7, 33-39 (1990).
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7. N. Sugihira and K. T. Suzuki, Biol. Trace Elem. Res. 29, 1-10 (1991). 8. E. Kobayashi, N. Sugihira, and K. T. Suzuki, Trace Elem. Med. 7, 114-117 (1990). 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
C. O. Lee, A. Taylor, and E. E. Windhager, Nature 287, 859-861 (1980). K. J. Ullrich, G. Rumrich, and S. Kloss, Pflugers Arch. 364, 223-228 (1976). P. Gmaj, H. Muter, and R. Kinne, Biochem. J. 178, 549-557 (1979). A. Jayakumar, L. Cheng, C. T. Liang, and B. Sacktor, J. Biol. Chem. 259, 10827-10833 (1984). P. Gmaj, H. Murer, and E. Carafoli, FEBS Lett. 144, 226-230 (1982). M. P. E. van Heeswijk, J. A. M. Geertsen, and C. H. van Os, J. Membrane Biol. 79, 19-31 (1984). K. W. Snowdowne and A. B. Borle, J. Biol. Chem. 260, 14998-15007 (1985). P. Gmaj, G. Bianchi, and H. Muter, Biochim. Biophys. Acta 941, 187-197 (1988). A. K. Mircheff and E. M. Wright, J. Membrane Biol. 28, 309-333 (1976). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265-275 (1951). B. A. Bulos and B. Sacktor, Anal. Biochem. 95, 62-72 (1979). N. Sugihira, E. Kobayashi, and K. T. Suzuki, Biol. Trace Elem. Res. 25, 79-88 (1990). C. H. van Os, Biochim. Biophys. Acta 906, 195-222 (1987). H. Porzig, in Handbook of Stable Strontium, S. C. Skoryna, ed., Plenum, New York, 1981, pp. 183-200. H. Guimaraes-Motta, M. P. Sande-Lemos, and L. de Meis, J. Biol. Chem. 259, 8699-8705 (1984). H. J. Schatzmann and F. F. Vincenzi, J. Physiol. 201, 369-395 (1969). P. Gmaj, M. Zurini, H. Murer, and E. Carafoli, Eur. J. Biochem. 136, 71-76 (1983). J. A. Holguin, Arch. Biochem. Biophys. 251, 9-16 (1986).
Biological Trace Element Research
Vol. 34, 1992
ATP-Dependent Strontium Uptake by Basolateral Membrane Vesicles from Rat Renal Cortex in the Absence or Presence of Calcium NAOKO SUGIHIRA,* YASUNOBU AOKI, AND KAZUO T. SUZUKI
National Institute for Environmental Studies, Tsukuba, /barak/305, Japan Received May 28, 1991; Accepted July 21, 1991
ABSTRACT ATP-dependent Sr 2+ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent S r 2 + uptake and C a 2 + uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for C a 2+ and S r 2 + uptake. The apparent Km and V,,~x of ATP-dependent Sr 2 + uptake were both higher than those for C a 2 + uptake. ATP-dependent S r 2 + uptake by BLM vesicles diminished in the presence of 0.1 I~M Ca 2+ and was more markedly inhibited by 1 laM C a 2 + . Hill plots of S r 2+ uptake data with and without 0.1 ~M C a 2 + showed that the cooperative behavior of S r 2 + uptake was not changed by C a 2 +. In the presence of 0.1 BM C a 2 +, the affinity of the transport system for Sr2+ and the velocity of S r 2 + uptake in the BLM were both decreased. However, the rate of C a 2+ uptake was not diminished by S r 2 + concentrations of < 1.6 p~M. These results suggest that C a 2 § is preferentially transported in the renal cortex BLM w h e n C a 2 + and S r 2 + are present at the same time. Index Entries Strontium; calcium; ATP-dependent uptake of cation; rat renal cortex; basolateral membrane vesicles; renal discrimination. *Author to w h o m all c o r r e s p o n d e n c e a n d reprint requests should b e addressed.
Biological Trace Element Research
45
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
46
E G T A , ethyleneglycol-bis-(f3-aminoethyl acid; HEDTA, N-hydroxyethylenediaminetetraacetic acid; (Mg2+/Ca2+)-ATPase, Mg2+-dependent Ca 2+ -stimulated adenosine triphosphatase; Mops, 4-morpholinepropanesulfonic acid; (Na +/K * )-ATPase, (Na +/K +)-stimulated adenosine triphosphatase. Abbreviations
ether)-N,N,N',N'-tetraacetic
INTRODOCTION The chemical properties of strontium (Sr) are very similar to those of calcium (Ca), and the metabolism and distribution of Sr in the body closely resemble those of Ca (1). However, discrimination of Sr in favor of Ca has been shown to occur during processes in which the animal body absorbs these elements from the environment and utilizes them (2-4). We have investigated renal discrimination between Sr and Ca during reabsorption in both experimental and diagnostic studies (5-8). Our previous observations suggested that renal tubules in rats (5-7) and humans (8) discriminate against Sr in favor of Ca by strictly regulating the reabsorption rate of Sr compared to that of Ca. However, the mechanisms by which the renal tubular cells discriminate between these two elements are not known. Although Ca reabsorption by the renal tubules is one of the major regulatory mechanisms for maintaining Ca homeostasis, its mechanisms have not been fully elucidated. It is generally assumed that Ca 2+ enters the tubular cells across the brush-border membrane from the tubular lumen by diffusion, because renal cytosolic free Ca 2§ concentrations are in the submicromolar range (9) and the membrane potential in the interior of the cell is negative. In contrast, C a 2 + has to be actively transported out of the cell across the basolateral segment of the plasma membrane, since the Ca 2+ electrochemical gradient at this membrane is reversed. Two different transport systems for C a 2 + in the basolateral membrane (BLM) have been proposed: One is a Na + / C a 2 + exchange process (10-12), and the other is ATP-dependent C a 2+ transport (11,13,14). ATP-dependent Ca 2+ transport has been shown to be mediated by a Ca2+-stimulated Mg2+-dependent ATPase in isolated BLM from rat renal cortex (13,14). van Heeswijk et al. (14) proposed that the (Mg2+/ Caa+)-ATPase system in rat renal cortex cells plays a primary role in Ca 2+ efflux. Snowdowne and Borle (15) reported that the Na +/Ca 2+ exchange is much less active than the ATPase system. There is a possibility that the active transport systems for Ca 2+ in renal BLM, described above, may be involved in the discrimination mechanisms between Sr and Ca in the renal tubules. In the present study, we examined ATP-dependent S r 2+ transport in BLM isolated from the renal cortex in the absence and presence of C a 2 + in order to find information that might help clarify the discrimination mechanisms between the two elements. Biological Trace Element Research
VoL 34, 1992
Renal Basolateral Membrane Sr2+ Transport
47
MATERIALS A N D METHODS Preparation of Renal Bl..a4 Vesicles
The renal cortices from six male Wistar rats (200-250 g) were used for each preparation. Basolateral membrane vesicles were isolated by a Percoll gradient procedure according to the method of Gmaj et al. (16). The isolated BLM were suspended in about I mL of a storage solution, which contained 100 mM mannitol, 100 mM KC1, 20 mM K'Mops (pH 7.0), 3 mM MgC12, 5 mM EGTA, I mM dithiothreitol, 1 mM ATP, and 15% (w/v) glycerol. The suspension was divided into 0.2-mL samples and stored in a liquid-nitrogen tank. Before each experiment, the membranes were thawed on ice, diluted to 1.5 mL with 100 mM KC1, 50 mM K-Mops (pH 7.4), 5 mM MgCI2, and 1 mM dithiothreitol, and centrifuged for 15 min. The washing step was repeated once, and the final membrane pellet was suspended in 0.3 mL of the same solution. Neither the ATP-dependent transport activity of Ca 2+ nor of Sr 2+ was changed by the freezing, thawing, and washing procedures. The activity of the ouabain-sensitive (Na +/K +)-ATPase, an enzyme marker for the BLM, was determined according to the method of Mircheff and Wright (17). Protein concentration was measured by the method of Lowry et al. (18) using bovine serum albumin as a standard. Ca 2 § or Sr 2 § Uptake Measurements
ATP-dependent uptake of Ca 2 + and Sr 2 + was determined according to the Ca 2+ uptake measurement method reported by Gmaj et al. (16). The kinetics of Ca 2+ and Sr 2+ transport was obtained from measurements of the initial rate of uptake for each ion (16). The incubation medium contained 100 mM KC1, 50 mM K-Mops (pH 7.4), 0.2 mM HEDTA, 0.5 mM EGTA, 10 b~M oligomycin, a calculated amount of MgC12 to keep the free Mg 2+ concentrations at about 4.7 mM, and calculated amounts of CaC12 or SrC12, or both. Finally, the medium contained 15 ~Ci 45Ca/mL and/or 2 p~Ci 85Sr/mL. The total amount of metal that had to be added to this system with several ligands and metal ions in order to reach the desired free metal ion concentration was calculated using the equation described by Bulos and Sacktor (19). The BLM suspension (20 b~L)was added to 100 b~L of the incubation medium, and the mixture was preincubated for 4 min at 37~ The uptake reaction was started by the addition of 5 ILL of 100 mM Mg-ATP to the medium. At given time intervals, 20-p,L samples were withdrawn and filtered through cellulose nitrate filters (0.45-p,m pore diameter). The filters were washed twice with 4-mL portions of ice-cold stopping solution containing 150 mM KC1, 20 mM K.Mops (pH 7.4), and 2 mM EGTA. The amount of radioactive 45Ca retained on the filters was measured in a liquid scintillation counter, and radioactive 85Sr was measured in a 3~ scintillation counter. The initial rates (v0) of Ca 2§ (or Sr 2+) uptake Biological Trace Element Research
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
48 =
6
e~
a.S
2
~
0
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,
I
0.2
0.0
,
I
0.4
,
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Free Ca 2, concentration (~tM) Fig. 1. Relationship between free C a 2+ concentration and the ATP-dependent C a 2+ uptake rate in basolateral membrane vesicles from rat kidney cortex. Each data point represents the mean + SE for three to seven observations in a single representative experiment. were calculated as the slopes of the regression lines of Ca 2+ (or Sr 2+) u p t a k e vs time.
RESULTS The specific activity of the ouabain-sensitive (Na +/K +)-ATPase, the e n z y m e m a r k e r for the BLM, in the BLM preparations was 1.37 + 0.13 p,mol Pi/min'mg protein (mean + SE, n = 3), indicating a 26-29-fold e n r i c h m e n t relative to the renal cortex h o m o g e n a t e . Ca 2 + u p t a k e from a m e d i u m containing 50 I~M CaCI2 a n d Sr 2 + u p t a k e from a m e d i u m containing 50 laM SrC12 w e r e both m e a s u r e d in BLM vesicles. In the presence of ATP, the BLM vesicles were f o u n d to accumulate Ca 2 + a n d Sr 2 § to levels exceeding those observed in the absence of ATP after 10 m i n of incubation by fourfold a n d eightfold, respectively. This indicates that A T P - d e p e n d e n t transport of S r 2 + as well as Ca 2 § occurs in the renal BLM. The kinetics of A T P - d e p e n d e n t Ca 2 + a n d Sr 2 + transport in the BLM vesicles is s h o w n in Figs. 1 a n d 2, respectively. Both Ca 2+ a n d Sr 2+ u p t a k e rates were increased d e p e n d i n g on cation concentration a n d were saturated at concentrations of cations h i g h e r than 0.35 a n d 1.25 t~M, respectively. The addition of 10 I~M vanadate, an inhibitor of (Mg 2+ / Ca~+)-ATPases, into the incubation m e d i u m r e d u c e d A T P - d e p e n d e n t Ca 2 + u p t a k e rates to 44-70% of those observed in the absence of vanadate. In the presence of 10 t~M vanadate, A T P - d e p e n d e n t Sr 2 + u p t a k e rates also d i m i n i s h e d to < 10% of those seen in the absence of vanadate. The relationship b e t w e e n free Sr 2+ concentration a n d ATPBiological Trace Element Research
VoL 34, 1992
Renal Basolateral Membrane Sr 2 + Transport
49
3 ~
8
O 6
~4
,
I
~
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1.0
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,
I
2.0
3.0
Free Sr 2§ concentration (~M) Fig. 2. Relationship between free Sr 2+ concentration and the ATP-dependent Sr 2 + uptake rate in basolateral membrane vesicles from rat kidney cortex. Each data point represents the mean _+ SE for three to six observations in a single representative experiment.
,~
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Free Sr 2+ concentration (~tM) Fig. 3. Relationship between free Sr 2§ concentration and the ATP-dependent Sr 2+ uptake rate in basolateral membrane vesicles from rat kidney cortex in the absence of Ca 2 + (C)) or in the presence of 0.1 pLM (m) or 1 p.M (&) free Ca 2+. Each data point represents the mean + SE for three to seven observations in a single representative experiment. d e p e n d e n t Sr 2 + u p t a k e in the presence of 0.1 ~M or 1 ~M free Ca 2 + is s h o w n in Fig. 3. The free Ca 2+ concentration in the cytosol of the proximal tubular e p i t h e l i u m is k n o w n to be approx 0.1 ~M (9). U n d e r this experimental condition, the ratios (w/w) of total Sr concentration to total Ca concentration in the reaction mixture were 0.01-1.63, w h i c h were about 10-100 times those observed in the b o d y (20). The presence of Biological Trace Element Research
Vol. 34, 1992
Sugihira, Aoki, and Suzuki
50
%
h...I
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-2
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-1
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Log [M 2+] Fig. 4. Hill plots of the kinetic data shown in Figs. 1-3. 0 - - 0 , ATPdependent C a 2+ uptake; 9169 ATPdependent Sr 2 + uptake in the absence of Ca 2 +; " - - ' , ATP-dependent Sr 2 + uptake in the presence of 0.1 ~M C a R+. [M 2+] represents the free cation concentration. The V,,,~xvalue used for these calculations was derived from the Lineweaver-Burk plot in the high ranges of cation concentration. 0.1 ~M Ca 2+ inhibited ATP-dependent Sr 2+ uptake compared with uptake observed in the absence of C a 2 + . The addition of 1 ~M C a 2 + further inhibited ATP-dependent Sr 2 + uptake. The data s h o w n in Figs. 1-3 were transformed to Hill plots (Fig. 4), since the Lineweaver-Burk reciprocal plots of the data were not completely linear (not shown). Sr 2+ uptake data in the presence of I ~M Ca 2+ were not transformed to a Hill plot, because the Sr ~+ uptake rate was not saturated in the concentration ranges examined (Fig. 3). The Vmaxvalues used for the calculations in the Hill plots were derived from the respective Lineweaver-Burk plots in the high-cation concentration range. All the Hill plots yielded straight lines (Ca 2+ uptake data, r = 0.80; Sr 2+ uptake data in the absence of Ca 2+, r = 0.94; Sr 2+ uptake data in the presence of 0.1 ~M Ca 2 +, r = 0.98). The slopes (Hill coefficients, nHil0 of the three lines were similar, in the range of 1.1-1.2. The kinetic parameters of ATP-dependent Ca 2 + uptake, and those of Sr 2 + uptake in the absence and presence of 0.1 ~M Ca 2 + are summarized in Table 1. The apparent Km (for the free metal ion) and Vmaxvalues of ATP-dependent Sr 2 + uptake in the absence of Ca 2 + were higher than those for C a 2+ uptake. The Km value for S r 2+ during ATP-dependent
Biological Trace Element Research
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Renal Basolateral Membrane Sr 2 + Transport
51
Table 1 Sr 2+ uptake ~'
Km (p,M) Wm a x (nmol/min 9 nni,
protein)
Ca 2+ uptake
- C a 2+
+ C a 2+
0.0548 +_ 0.0157
0.871 _ 0.344
1.99 _ 0.28
4.66 +_ 0.39 1.16 +_ 0.07
12.9 + 4.0 1.23 + 0.07
7.02 + 0.30 1.10 _+ 0.02
Table 2
S r 2+
C a 2+ uptake rate, nmol/min 9 mg protein
concentration, p.M 0 0.4 0.8 1.6 3.2 6.4 10.0
2.60 1.78 2.50 1.66 1.26 0.756 0.695
+ 0.17 _+ 0.05 +_ 0.12 + 0.20 b _+ 0.26 ~' _+ 0.062 ~' +_ 0.01if'
Table 3
Sr 2+ concentration, ~M 0 0.8 1.6 3.2 6.4 10.0 20.0
C a 2+ uptake rate, nmol/min 9 protein
3.16 3.54 3.26 3.28 2.59 2.92 2.12
+ 0.44 + 0.16 + 0.19 +- 0.31 _+ 0.05 _+ 0.23 +_ 0.25
Sr 2+ u p t a k e increased, a n d the V,~ x d e c r e a s e d w i t h the a d d i t i o n of 0.1 p,M Ca 2 +. The effects of Sr 2 + o n A T P - d e p e n d e n t Ca 2 + u p t a k e b y BLM vesicles in the p r e s e n c e of 0.1 p,M Ca 2+ w e r e also e x a m i n e d (Table 2). The Ca 2 + u p t a k e rate w a s n o t affected b y Sr 2 + at c o n c e n t r a t i o n s l o w e r t h a n 1.6 laM, b u t it w a s i n h i b i t e d b y Sr at c o n c e n t r a t i o n s h i g h e r t h a n a n d equal to 1.6 ~M. The effects of Sr 2 + o n A T P - d e p e n d e n t Ca z§ u p t a k e in t h e p r e s e n c e of I ~ M free Ca 2+ are s h o w n in Table 3. T h e ATPd e p e n d e n t Ca 2 + u p t a k e rate w a s n o t c h a n g e d by Sr 2+ at a n y of the c o n c e n t r a t i o n s t e s t e d (-< 20 ~M).
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Sugihira, Aoki, and Suzuki
DISCUSSION The basolateral segment of the renal tubular plasma membrane possesses an active transport system for Ca 2+ (10,11,13,14,21). This mechanism is thought to regulate the intracellular Ca 2+ concentration and to be involved in Ca reabsorption by the renal tubules (10). BLM vesicles isolated from rat renal cortex have also been reported to have ATP-dependent uptake of Ca 2+ (11,13,14). However, no mechanism of Sr reabsorption by the renal tubule or of discrimination between Sr and Ca during the reabsorption step had been elucidated. Strontium, unlike other divalent cations, has been shown to share a number of active membrane transport systems (22). For example, Sr 2+ has been shown to accumulate in vesicles derived from the sarcoplasmic reticulum of skeletal muscle at the expense of ATP hydrolysis as well as Ca 2§ uptake (23). Human red cells also have an ATP-dependent ion pump that transports not only Ca 2+, but also Sr 2. (24). The operation of the pump is also connected to the activity of a transport ATPase (22). In the present study, ATP-dependent uptake of Sr ~+, as well as that of Ca 2+, was observed in BLM vesicles derived from the renal cortex. This Sr 2+ uptake system is highly sensitive to vanadate. Vanadate is known to inhibit ATP-dependent Ca 2+ transport and (Mg2+/Ca 2+) ATPase activities in BLM from rat renal cortex (13,25)9 These findings suggest the possibility that Sr 2+ was taken up into the BLM vesicles by the same mechanisms as Ca 2§ In the presence of 0.1 ~M Ca 2+, the ATP-dependent Sr 2+ uptake rate was inhibited (Fig. 3). Hill plots of these data indicate similarities in the kinetic behavior of Ca 2 + and Sr 2 + uptake, and in Sr 2+ uptake in the presence and absence of 0.1 ~M Ca 2 + (Fig. 4). These results suggest that the cooperative behaviors of ATP-dependent C a 2 + and S r 2 + transport in BLM are similar and that the inhibition of ATP-dependent Sr 2+ transport by 0.1 ~M C a 2+ is not an allosteric effect. Kinetic parameters obtained for ATP-dependent Ca 2+ uptake by BLM vesicles were in accordance with those previously published (14,25). The K,, of ATP-dependent S r 2+ uptake was higher than that of the Ca 2+ uptake (Table 1), indicating the lower affinity of the BLM transport system for S r 2 +. These observations in the BLM vesicles from rat renal cortex are in agreement with those reported for an ATPase in the sarcoplasmic reticulum, the affinity of which was 12-14 times higher for Ca 2+ than for Sr 2§ (26). The V,,a~ for ATPase activities in skeletal sarcoplasmic reticulum vesicles was shown to be higher in the presence of Sr z + than in the presence of Ca 2+ (26). The author suggested that this could be explained by a lower affinity of the internal binding sites on the ATPase for Sr 2+ as compared to their affinity for C a 2+ and that this decreases feedback inhibition of the ATPase in sarcoplasmic reticulum vesicles (23). The higher Vmaxof ATP-dependent S r 2 + uptake compared
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Renal Basolateral Membrane Sr 2 . Transport
53
to that of Ca 2 + uptake that we obtained (Table 1) might also be explained by this mechanism. In erythrocyte ghosts, Ca 2 + is transported almost exclusively w h e n C a 2 + and Sr 2 + are present at the same time (24). In our results, ATPd e p e n d e n t S r 2 + uptake in BLM vesicles from the renal cortex was diminished by 0.1 ~M Ca 2§ and was even more markedly inhibited by 1 ~M Ca 2 + (Fig. 3). In the presence of 0.1 I~M Ca 2 +, the apparent Km of Sr 2 + uptake increased compared with that observed in the absence of Ca 2 § (Table 1). In addition to this, the Vmax of Sr 2 + uptake was diminished by the presence of 0.1 ~M Ca 2 +, which may be owing to feedback inhibition of the ATPase by Ca 2 4, possibly by the mechanism described above. We observed that 0.1 ~M free Ca 2 + , which is identical to the estimated cytosolic free Ca 2+ concentration in kidney cells, decreases the affinity of Sr 2 § for the transport system and the velocity of Sr 2 + uptake in the BLM. In the experiment in which the incubation m e d i u m contained 0.1 ~M Ca 2+ and various concentrations of free Sr 2+, the ATP-dependent Ca 2+ uptake rate was not changed by Sr 2+ concentrations of ~ 1.6 ~M (Table 2). In the presence of a higher concentration of Ca 2+ (1 ~M), the ATP-dependent Ca 2 § uptake rate was not decreased by Sr 2 § at any of the concentrations examined (Table 3). Our results suggest that Ca 2+ is preferentially transported in the BLM from rat renal cortex w h e n Ca 2 + and Sr 2 + are present at the same time, even though the ratios (w/w) of total Sr concentration to total Ca concentration in the reaction mixture are 10-100 times those observed in the body. It is likely that this preferential transport of Ca 2 + in BLM may contribute to the discrimination mechanisms between Sr and Ca during the reabsorption process along the proximal tubule.
ACKNOWLEDGMENT We wish to thank Dr. M. Murakami for his encouragement.
REFERENCES 1. R. H. Wasserman, E. M. Romney, M. W. Skougstad, and R. Siever, in Geochemistry and the Environment. vol. 2, National Academy of Science, Washington, D.C., 1977, pp. 73--87. 2. G. W. Dolphin and I. S. Eve, Phys. Med. Biol. 8, 193-203 (1963). 3. C. L. Comar and R. H. Wasserman, in Mineral Metabolism: An Advanced Treatise, C. L. Comar and F. Bronner, eds., Academic, New York, 1964, pp. 523-572. 4. C. L. Comar, in Strontium Metabolism, J. M. A. Lenihan, J. F. Loutit, and J. H. Martin, eds., Academic, New York, 1967, pp. 17-31. 5. N. Sugihira and K. T. Suzuki, Biol. Trace Elem. Res. 22, 71-82 (1989). 6. N. Sugihira and K. T. Suzuki, Trace Elem. Med. 7, 33-39 (1990).
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7. N. Sugihira and K. T. Suzuki, Biol. Trace Elem. Res. 29, 1-10 (1991). 8. E. Kobayashi, N. Sugihira, and K. T. Suzuki, Trace Elem. Med. 7, 114-117 (1990). 9. 10. 11. 12.
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C. O. Lee, A. Taylor, and E. E. Windhager, Nature 287, 859-861 (1980). K. J. Ullrich, G. Rumrich, and S. Kloss, Pflugers Arch. 364, 223-228 (1976). P. Gmaj, H. Muter, and R. Kinne, Biochem. J. 178, 549-557 (1979). A. Jayakumar, L. Cheng, C. T. Liang, and B. Sacktor, J. Biol. Chem. 259, 10827-10833 (1984). P. Gmaj, H. Murer, and E. Carafoli, FEBS Lett. 144, 226-230 (1982). M. P. E. van Heeswijk, J. A. M. Geertsen, and C. H. van Os, J. Membrane Biol. 79, 19-31 (1984). K. W. Snowdowne and A. B. Borle, J. Biol. Chem. 260, 14998-15007 (1985). P. Gmaj, G. Bianchi, and H. Muter, Biochim. Biophys. Acta 941, 187-197 (1988). A. K. Mircheff and E. M. Wright, J. Membrane Biol. 28, 309-333 (1976). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265-275 (1951). B. A. Bulos and B. Sacktor, Anal. Biochem. 95, 62-72 (1979). N. Sugihira, E. Kobayashi, and K. T. Suzuki, Biol. Trace Elem. Res. 25, 79-88 (1990). C. H. van Os, Biochim. Biophys. Acta 906, 195-222 (1987). H. Porzig, in Handbook of Stable Strontium, S. C. Skoryna, ed., Plenum, New York, 1981, pp. 183-200. H. Guimaraes-Motta, M. P. Sande-Lemos, and L. de Meis, J. Biol. Chem. 259, 8699-8705 (1984). H. J. Schatzmann and F. F. Vincenzi, J. Physiol. 201, 369-395 (1969). P. Gmaj, M. Zurini, H. Murer, and E. Carafoli, Eur. J. Biochem. 136, 71-76 (1983). J. A. Holguin, Arch. Biochem. Biophys. 251, 9-16 (1986).
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