Brain Research, 566 (1991) 127-130 (~) 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/91/$03.50
127
BRES 17213
Effect of cadmium
o n C a 2+
Jyotsna
transport in brain microsomes
Shah 1 and Harish
C. Pant 2
ZDepartment of Biophysics, Boston University School of Medicine, Boston, MA 02118 (U.S.A.) and ZLaboratory of Neurochemistry, National Institute of Neurological and Communicative Disease and Stroke, NIH, Bethesda, MD 20892 (U.S.A.)
(Accepted 16 July 1991) Key words: Cadmium; Brain microsome; Intracellular calcium; Release; Sulfhydryl reagent
The effect of Cd2÷ on Ca2+ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using 45Ca2+. Cadmium ion (Cd 2+) shows a dose-dependent inhibition of Ca2+ -ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca 2+ uptake. In addition to this, Cd2+ also stimulates a rapid release of Ca 2+ (tv2 - 0.5 min) from the microsomes in a dose-dependent manner. The effect of Cd 2+ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd 2+ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTr suggesting a role of sulfhydryl groups in Ca2+-transport.
INTRODUCTION Cadmium ion (Cd 2+) can induce cardiovascular, testicular, and renal tubular lesions in humans when consumed continuously from food and water. Previously, it has been demonstrated that long-term administration of Cd 2÷ to rats and rabbits causes persistent hypertension. The hypertension resulting from Cd 2+ was reversed by injection of a chelator 7's'n. Perry et al. 5 and Thind et al. ts reported that Cd 2+ applied in vitro to aortic strips produced decreased responsiveness to norepinephrine, epinephrine, angiotensin and potassium ion (K÷). These authors postulated that this inhibitory effect could be associated with binding of Cd 2+ by sulfhydryl groups of contractile proteins. Calcium contractures observed in aortas depolarized by K + (in Ca2+-free media) were also inhibited by Cd 2+ (ref. 14). Sumida et al. tl studied the effect of divalent cations on the ATP-dependent calcium uptake by microsomes from bovine aortic smooth muscle and found that Cd 2+ inhibited both the Ca 2+ uptake as well as formation of the phosphorylated intermediate enzyme (E-P). This effect was inhibited by thiol-reagents. The addition of micromolar concentrations of heavy metals, e.g. Ag + and Fig2+ induced rapid Ca 2+ release from Ca2+-loaded sarcoplasmic reticulum (SR) vesicles and the extent of Ca 2+ release was dependent on the binding of the metal to sulfhydryl group(s) on an SR protein t. In a later study, Salama et al. ~ reported that Ag + ions reacted with a protein or proteins in the SR, probably not the Ca 2+, Mg2+-ATPase, to induce a rapid
release of Ca 2+, from a physiological site. However, most striking results were shown in reconstituted vesicles containing the Ca 2+, Mge+-ATPase (purified from SR) as the only protein 3. Ag + induced a rapid release of Ca z+ from such reconstituted vesicles and, also, had a marked inhibitory effect on the ATPase activity of the purified ATPase. On the basis of these results, it was concluded that the Ca 2+, Mg2+-ATPase can act as a pathway for rapid Ca 2÷ release from SR. In our previous studies, we characterized Ca 2÷ transport properties of the microsomes isolated from the rat brain 9'~°, Here we have focussed on the possible involvement of heavy metal ions (e.g. Cd 2÷ and Ag +) on Ca 2÷ transport in brain microsomes and attempts are made to gain an insight into the mechanism of these effects in the central nervous system. MATERIALS AND METHODS Most chemicals and reagents used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.), or Fisher Scientific (Pittsberg, PA, U.S.A.) in highest purity grade offered. 4S CaCl2 and [7"32P]ATPwere obtained from New England Nuclear. Brain microsomes were prepared from male Sprague-Dawley rats (150-200 g) by the method previously published9. Ca2+ uptake and release were determined by measuring the radioactive Caz+ trapped inside the vesicles. To determine the uptake, microsomes resuspended in buffer A (containing 100 mM KCI, 20 mM HEPES, 2.5 mM MgC!2, 0.1 mM EGTA and 101 l~M CaCIz, pH 7.0), were preincubated in the same buffer with 0.1 ~Ci 4SCaCle/ml (1 mg/ml of protein) at 37 °C for 4 min. The calculated final free Caz+ in the solution was 5/~M2. Cae+ uptake was initiated by the addition of 1.6 mM ATP to the incubation media. Samples were removed after the specific time intervals. The reac-
Correspondence: J. Shah, Department of Biophysics, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118, U.S.A.
128 tion was terminated b~ the addition of 20 vols. of ice-cold reaction buffer devoid of 4.Ca..÷ and filtered immediately using a cell harvester M-24B (Brandel). Filters were washed three times using 5 m] of buffer each time and counted in a Beckman LS-8100 Liquid Scintillation Counter. To measure the Ca2+-release, microsomes were incubated in buffer A in the presence of 0. l ,uCi "SSCaCla/mland 1.6 mM ATP for 15 min at 37 °C. After 15 min incubation, desired amount of Cd2+ was added and samples were removed at specific time intervals. Radioactivity was measured as described above. Calcium ATPase activity was determined by measuring the 32p inco:poration in the presence and absence of Cd2+. The microsomes (100 ~g protein) were incubated in KCI medium (in the presen,:e of ouabain) for 20 min at 37 °C prior to addition of D,-32p]ATP (500,000 cpm approximately) and then incubated for another 30 min in the presence of radioactive ATP. Reaction was terminated by the addition of freshly made ice-cold 12% (w/v) activated charcoal in 0.8% (w/v) SDS solution. Tubes were mixed well and kept in ice for 10 min. This mixture was centrifuged at 12,000 g for 20 min and the supernatant was decanted into precooled tubes. Aliquots of supernatant were added to ACS scintillant and counted for radioactivity. The net activity was determined as the difference in the rate of reaction in the presence and absence of Na-orthovanadate. Protein was measured by the method of Lowry etal. using bovine serum albumin as standard ~. RESULTS AND DISCUSSION
Effect of Ca¢+ on ATP.dependent Ca 2+ accumulation Calcium uptake was measured as described in Materials and Methods. Fig, I shows the 4SCa'+-uptake in the presence and absence of Cd 2+. In the control experiments, addition of 1,6 mM ATP to the microsomes (proincubated in buffer A) results in an exponential uptake of calcium (ttr~ " 1-1.5 min) which attains a steady state level in S-10 rain (Fig, 1), Presence of 100 pM Cd 2÷ reduces the total uptake to 60% without any significant
change in tlr~ value (Fig. 1). ~this inhibition of Ca2+-up take is dependent upon Cd 2+ concentration and is almost completely blocked at higher concentration of Cd 2+, e.g. 300 pM (data not shown). To examine if this observed reduction in the radioactive Ca 2+ uptake is due to inhibition of Ca2+-ATPase activity, we studied the effect of Cd 2+ on Cae+-ATPase activity, cae+-ATPase activity was measured as described in Materials and Methods. Fig. 2 shows the dependence of the mi.crosomal Ca2+-ATPase activity as a function of Cd 2+ concentration. A half-maximal inhibition of Ca 2+ATPase activity was seen at 100 pM Cd 2+ concentration and maximal inhibition occurred at 250 pM Cd 2+ concentration. These data show that Cd 2+ inhibits both the processes (Ca2+-uptake and ATPase activity) to the same extent (compare Figs. 1 and 2), suggesting that Cd 2+ reduces the ATP-dependent Ca 2+ uptake by inhibiting the Ca2+-ATPase activity.
Cd2+.induced Ca2+ release Fig. 3A shows the effects of Cd 2+ on Ca2+-loaded microsomes. The data indicate that Cd 2+ induces a rapid release of Ca2+ (tlr~ = 0.5 min) and almost 50% of total accumulated Ca 2÷ is released by 100 pM Cd 2+ and approximately 80% Ca 2+ is released at 250 pM concentration of Cd 2+ after which no further increase is observed.
Fig. 3B shows the dose dependency of Cd2+-induced Ca 2+ release, In order to examine any correlation between the Cd2+-induced inhibition of Ca 2+ uptake and Cd2+-in duced release of Ca 2÷, we compared the effect of Naorthovanadate (which is a potent Ca2+-ATPase inhibitor) and Cd :+ (Fig, 3A) on the Ca2+-loaded microsomes. "~'he microsomes are loaded with Ca 2+ and after steadystate level is obtained I00 pM Cd 2+ or I00 pM Na-orthovanadate is added to the reaction mixture and sam-
Z lo
100
D
0
5
10
15
TIME (rain) Fig, l, Effect of Cd 2÷ on Mg'+-ATP.dependent Caa+ uptake, Microsomal fractions (1 mg/ml protein) were preincubated in the re. action mixture containing I00 mM KCI, 20 mM HEPES, 2,5 mM MgCI.~, 5 ~M free Ca2÷, 0, l/~Ci/ml 4SCaCl2, (pH 7,0), in the presence (c3) and absence (e) of 100 ltM Cd2+ at 37 °C for 4 rain, Uptake was initiated by the addition of 1,6 mM ATP into the, reaction mixture and aliquots of 200 ld were removed at specific time intervals, Reaction was terminated by addition of 20 vols, of ice-cold buffer devoid of '~SCa2+ and filtered immediately, Filters wove washed 3 times with ti~e same buffer and counted for radioactivity in a liquid scintillation counter. Curve represented by triangles (A) shows the '~SCa2+ accumulation in the absence of ATE
20
I I00
I 2OO
i 3O0
Cd~- CONC. I~MI Fig. 2. Effect of Cd 2+ on Ca2+-ATPase activity. Ca2+-ATPase activity was measured as described in Materials and Methods. Data presentec~ are an average of 3 experiments.
129 pies are removed every 10 s. Radioactivity was measured as described in Materials and Methods. It can be seen from Fig. 3 that vanadate does not stimulate Ca2+-release in a way similar to Cd 2+ (i.e. does not show a sudden Ca 2+ release). However, a slow diffusion of Ca 2+ appeared in the presence of vanadate (possibly due to inhibition of Ca 2+ reuptake). Since vanadate does not cause a sudden release of Ca 2+ (although it definitely inhibits Ca2+-ATPase activity), this indicates that the ability of Cd 2+ to inhibit Ca2+-ATPase activity may not be responsible for the Cd2+-induced Ca 2+ release and probably involves some other mechanism. In order to investigate the mechanism of Cd2+-in duced Ca 2+ release, the effect of sulfhydryl (SH) binding agents, e.g. D T r (which was effective in case of bovine aortic smooth muscle microsomes and sarcoplasmic reticulum), is studied on Cd2+-induced effects. First the effect of these SH reagents is studied on the Ca2+-trans port. For this, 1 mM cysteine or 1 mM DTT is added to the Ca2+-loaded microsomes and the experiment is performed as described previously. It is found that cysteine or DTT alone do not have any effect on Ca 2+ transport
A
~,0
Cd~*
|
I
i
10
12
14
ii ii
i
]
i
18 18 TIME (mln|
I
I
20
22
(data not shown). To examine the effect of Cd 2+ in the presence of SH binding reagents, the Ca2+-loaded microsomes were preincubated with 1 mM cysteine or 1 mM dithiothreitol (DTT) prior to exposure to 100/~M C d 2+. F i g . 4 s h o w s that Cd2+-induced C a 2+ release is reduced to 47% in the presence of cysteine or DTT. In conclusion, the effect of cadmium-ions is examined on the Ca2+-transport properties in the brain microsomes. The data presented here clearly demonstrate that: (i) Cd 2+ acts as Ca2+-ATPase inhibitor and this inhibition can be reversed by DTT; and (ii) C d 2+ induces Ca2+-release which can be partially inhibited if thiol reagents were added to the microsomes prior to the Cd 2+ exposure. From our results, we suggest that Cd 2+ ions exert their effect by directly interacting with Ca 2+ATPase (to reduce the Ca2+..ATPase activity and consequently, decrease Ca 2+ uptake) in agreement with the results of Sumida et al.l~. In a previous publication by Gould et ai. 3, it has been shown that in reconstituted vesicles containing the Ca2+,Mg2+-ATPase as the only protein, the ATPase can act as a pathway for Ag+-in duced Ca 2+ efflux. Unfortunately, from our results with the microsomes, we car, not predict whether the stimulatory effect of Cd 2+ is also induced by interaction with the same protein, i.e. Ca2+-ATPase (as was suggested by Gould et al. 3 in SR) or different proteins are involved in the two processes. However, since both of these effects are reversed by thiol reagents (which maintain the sulphur-containing residues in the reduced state), these results may be attributed to oxidation of certain SH residues by Cd 2+, the reduced form/forms of which is/are essential for the enzyme (Ca2+-ATPase activity) function or Ca 2+ channel regulatory function.
!-
8O
o
X I
100 2OO 3OO 4OO Cd2+ CONCENTRATION (/AM) Fig. 3. CdZ+-induced Ca2+ release. Microsomes (I mg/ml protein)
were inr,ubated with buffer containing 100 mM KCI, 2.5 mM MgCI2, 5/~M free Ca2+, 20 mM HEPES, 0.1/~Ci 45Ca2+ and 1.6 mM ATP (pH 7.0). After 15 min incubation at 37 °C, different concentrations of C d 2+ were added to the reaction mixture and 200/~1 aliquots were removed at 10 s interval. Radioactivity was measured as described in Fig. 1. (A) Ca 2+ release at various Cd 2+ concentrations. Curve presented by broken line shows Ca2+-release by 100 /~M Na.orthovanadate. The data represented are typical of 5 experiments. (B) Dose d e p e n d e n c y of Cd 2+ -induced Ca 2+ release.
10 8
a. (J
01
I
12
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14
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I
16 18 20 TIME (min)
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22
Fig. 4. Effect of cysteine on Cd2+-induced Ca2+ release: Cd2+-in -
duced Ca 2+ release was measured in the presence (O) and absence (o) of 1 mM cysteine. Cd 2+ concentration was 250/~M.
130 REFERENCES 1 Abramson, J.J., Trimm, J,L., Weden, L. and Salama, O., Heavy metal induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle, Proc. Natl. Acad. Sci, U.S.A.. 80 (1983) 1526-1532. 2 Fabiato, A. and Fabiato, F., Calculator programme for computing the compositions of solutions containing multiple metals and ligands used for experiments inskinned muscle cells, J. Physiol. (Paris), 75 (1979) 463-505. 3 Gould, G.W., Colyer, J., East, J.M. and Lee, A.G., Silver ions trigger Ca2÷ release by interaction with the (Ca2+-Mg2+)ATpase in reconstituted systems, J. Biol. Chem., 262 (1987) 7676-7679. 4 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 5 Perry, H.M., Schoepfle, E. and Bourgoignie, J., In vitro production and inhibition of aortic vasoconstriction by mercuric. cadmium and other metal ions, Proc. Sac. Exp. Biol. Med., ;74 (1967) 485-490. 6 Salama, G. and Abramson, J.. Silver ions trigger Ca 2+ release by acting at the apparent physiological site in sarcoplasmic reticulum site, J. Biol. Chem.. 259 (1984) 13363-13369. 7 Schroder, H.A., Kroll. S.A., Little, J.W.. Livingston, P.O. and Myers, M.A.G., Hypertension in rats from injection of cadmium, Arch. Environ. Health, 13 (1966) 788-789.
8 Schroder, H.A., Nason, A.E, Prior, R.E., Reed, J.B. and Haessler, W.T., Influence of cadmium on renal ischemic hypertension in rats, Am. I. Physiol., 214 (1968) 469-474. 9 Shah, J., Cohen, R.S. and Pant, H.C., Inositol trisphosphate induced calcium release in rat brain microsomes, Brain Research, 612 (1987) 1-6. 10 Shah, J. and Pant, H.C., Potassium channel blockers inhibit inositol trisphosphate induced calcium release in the microsomal fractions isolated from the rat brain, Biochem. l., 250 (1988) 617. 11 Sumida, M., Hamada, M., Takenaka, H., Hirata, Y., Nishigauchi, K. and Okuda, H., Ca2+,Mg2+-ATPase of microsomal m~mbranes from bovine aortic smooth muscle: effects of Sr2+ and C d 2+ o n C a 2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATpase, I. Biochem., 100 (1986) 765-772. 12 Thind, G.S,, Karreman, G., Stephan, K.F. and Blackmore, W.S., Vascular reactivity and mechanical properties of normal and cadmium-hypertensive rabbits, l. Lab. CIin, Med., 76 (1970) 560-568. 13 Thind, G.S., Stephan, K.E and Blackmore, W.S,, Inhibition of vasopressor responses by cadmium, Am, J. Physiol,, 219 (1970) 577-583. 14 Toda, N., Influence of cadmium ions on contractile response of isolated aortas to 3timulatory agents, Am, J. Physiol., 225 (1973) 350-355.
127
BRES 17213
Effect of cadmium
o n C a 2+
Jyotsna
transport in brain microsomes
Shah 1 and Harish
C. Pant 2
ZDepartment of Biophysics, Boston University School of Medicine, Boston, MA 02118 (U.S.A.) and ZLaboratory of Neurochemistry, National Institute of Neurological and Communicative Disease and Stroke, NIH, Bethesda, MD 20892 (U.S.A.)
(Accepted 16 July 1991) Key words: Cadmium; Brain microsome; Intracellular calcium; Release; Sulfhydryl reagent
The effect of Cd2÷ on Ca2+ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using 45Ca2+. Cadmium ion (Cd 2+) shows a dose-dependent inhibition of Ca2+ -ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca 2+ uptake. In addition to this, Cd2+ also stimulates a rapid release of Ca 2+ (tv2 - 0.5 min) from the microsomes in a dose-dependent manner. The effect of Cd 2+ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd 2+ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTr suggesting a role of sulfhydryl groups in Ca2+-transport.
INTRODUCTION Cadmium ion (Cd 2+) can induce cardiovascular, testicular, and renal tubular lesions in humans when consumed continuously from food and water. Previously, it has been demonstrated that long-term administration of Cd 2÷ to rats and rabbits causes persistent hypertension. The hypertension resulting from Cd 2+ was reversed by injection of a chelator 7's'n. Perry et al. 5 and Thind et al. ts reported that Cd 2+ applied in vitro to aortic strips produced decreased responsiveness to norepinephrine, epinephrine, angiotensin and potassium ion (K÷). These authors postulated that this inhibitory effect could be associated with binding of Cd 2+ by sulfhydryl groups of contractile proteins. Calcium contractures observed in aortas depolarized by K + (in Ca2+-free media) were also inhibited by Cd 2+ (ref. 14). Sumida et al. tl studied the effect of divalent cations on the ATP-dependent calcium uptake by microsomes from bovine aortic smooth muscle and found that Cd 2+ inhibited both the Ca 2+ uptake as well as formation of the phosphorylated intermediate enzyme (E-P). This effect was inhibited by thiol-reagents. The addition of micromolar concentrations of heavy metals, e.g. Ag + and Fig2+ induced rapid Ca 2+ release from Ca2+-loaded sarcoplasmic reticulum (SR) vesicles and the extent of Ca 2+ release was dependent on the binding of the metal to sulfhydryl group(s) on an SR protein t. In a later study, Salama et al. ~ reported that Ag + ions reacted with a protein or proteins in the SR, probably not the Ca 2+, Mg2+-ATPase, to induce a rapid
release of Ca 2+, from a physiological site. However, most striking results were shown in reconstituted vesicles containing the Ca 2+, Mge+-ATPase (purified from SR) as the only protein 3. Ag + induced a rapid release of Ca z+ from such reconstituted vesicles and, also, had a marked inhibitory effect on the ATPase activity of the purified ATPase. On the basis of these results, it was concluded that the Ca 2+, Mg2+-ATPase can act as a pathway for rapid Ca 2÷ release from SR. In our previous studies, we characterized Ca 2÷ transport properties of the microsomes isolated from the rat brain 9'~°, Here we have focussed on the possible involvement of heavy metal ions (e.g. Cd 2÷ and Ag +) on Ca 2÷ transport in brain microsomes and attempts are made to gain an insight into the mechanism of these effects in the central nervous system. MATERIALS AND METHODS Most chemicals and reagents used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.), or Fisher Scientific (Pittsberg, PA, U.S.A.) in highest purity grade offered. 4S CaCl2 and [7"32P]ATPwere obtained from New England Nuclear. Brain microsomes were prepared from male Sprague-Dawley rats (150-200 g) by the method previously published9. Ca2+ uptake and release were determined by measuring the radioactive Caz+ trapped inside the vesicles. To determine the uptake, microsomes resuspended in buffer A (containing 100 mM KCI, 20 mM HEPES, 2.5 mM MgC!2, 0.1 mM EGTA and 101 l~M CaCIz, pH 7.0), were preincubated in the same buffer with 0.1 ~Ci 4SCaCle/ml (1 mg/ml of protein) at 37 °C for 4 min. The calculated final free Caz+ in the solution was 5/~M2. Cae+ uptake was initiated by the addition of 1.6 mM ATP to the incubation media. Samples were removed after the specific time intervals. The reac-
Correspondence: J. Shah, Department of Biophysics, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118, U.S.A.
128 tion was terminated b~ the addition of 20 vols. of ice-cold reaction buffer devoid of 4.Ca..÷ and filtered immediately using a cell harvester M-24B (Brandel). Filters were washed three times using 5 m] of buffer each time and counted in a Beckman LS-8100 Liquid Scintillation Counter. To measure the Ca2+-release, microsomes were incubated in buffer A in the presence of 0. l ,uCi "SSCaCla/mland 1.6 mM ATP for 15 min at 37 °C. After 15 min incubation, desired amount of Cd2+ was added and samples were removed at specific time intervals. Radioactivity was measured as described above. Calcium ATPase activity was determined by measuring the 32p inco:poration in the presence and absence of Cd2+. The microsomes (100 ~g protein) were incubated in KCI medium (in the presen,:e of ouabain) for 20 min at 37 °C prior to addition of D,-32p]ATP (500,000 cpm approximately) and then incubated for another 30 min in the presence of radioactive ATP. Reaction was terminated by the addition of freshly made ice-cold 12% (w/v) activated charcoal in 0.8% (w/v) SDS solution. Tubes were mixed well and kept in ice for 10 min. This mixture was centrifuged at 12,000 g for 20 min and the supernatant was decanted into precooled tubes. Aliquots of supernatant were added to ACS scintillant and counted for radioactivity. The net activity was determined as the difference in the rate of reaction in the presence and absence of Na-orthovanadate. Protein was measured by the method of Lowry etal. using bovine serum albumin as standard ~. RESULTS AND DISCUSSION
Effect of Ca¢+ on ATP.dependent Ca 2+ accumulation Calcium uptake was measured as described in Materials and Methods. Fig, I shows the 4SCa'+-uptake in the presence and absence of Cd 2+. In the control experiments, addition of 1,6 mM ATP to the microsomes (proincubated in buffer A) results in an exponential uptake of calcium (ttr~ " 1-1.5 min) which attains a steady state level in S-10 rain (Fig, 1), Presence of 100 pM Cd 2÷ reduces the total uptake to 60% without any significant
change in tlr~ value (Fig. 1). ~this inhibition of Ca2+-up take is dependent upon Cd 2+ concentration and is almost completely blocked at higher concentration of Cd 2+, e.g. 300 pM (data not shown). To examine if this observed reduction in the radioactive Ca 2+ uptake is due to inhibition of Ca2+-ATPase activity, we studied the effect of Cd 2+ on Cae+-ATPase activity, cae+-ATPase activity was measured as described in Materials and Methods. Fig. 2 shows the dependence of the mi.crosomal Ca2+-ATPase activity as a function of Cd 2+ concentration. A half-maximal inhibition of Ca 2+ATPase activity was seen at 100 pM Cd 2+ concentration and maximal inhibition occurred at 250 pM Cd 2+ concentration. These data show that Cd 2+ inhibits both the processes (Ca2+-uptake and ATPase activity) to the same extent (compare Figs. 1 and 2), suggesting that Cd 2+ reduces the ATP-dependent Ca 2+ uptake by inhibiting the Ca2+-ATPase activity.
Cd2+.induced Ca2+ release Fig. 3A shows the effects of Cd 2+ on Ca2+-loaded microsomes. The data indicate that Cd 2+ induces a rapid release of Ca2+ (tlr~ = 0.5 min) and almost 50% of total accumulated Ca 2÷ is released by 100 pM Cd 2+ and approximately 80% Ca 2+ is released at 250 pM concentration of Cd 2+ after which no further increase is observed.
Fig. 3B shows the dose dependency of Cd2+-induced Ca 2+ release, In order to examine any correlation between the Cd2+-induced inhibition of Ca 2+ uptake and Cd2+-in duced release of Ca 2÷, we compared the effect of Naorthovanadate (which is a potent Ca2+-ATPase inhibitor) and Cd :+ (Fig, 3A) on the Ca2+-loaded microsomes. "~'he microsomes are loaded with Ca 2+ and after steadystate level is obtained I00 pM Cd 2+ or I00 pM Na-orthovanadate is added to the reaction mixture and sam-
Z lo
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0
5
10
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TIME (rain) Fig, l, Effect of Cd 2÷ on Mg'+-ATP.dependent Caa+ uptake, Microsomal fractions (1 mg/ml protein) were preincubated in the re. action mixture containing I00 mM KCI, 20 mM HEPES, 2,5 mM MgCI.~, 5 ~M free Ca2÷, 0, l/~Ci/ml 4SCaCl2, (pH 7,0), in the presence (c3) and absence (e) of 100 ltM Cd2+ at 37 °C for 4 rain, Uptake was initiated by the addition of 1,6 mM ATP into the, reaction mixture and aliquots of 200 ld were removed at specific time intervals, Reaction was terminated by addition of 20 vols, of ice-cold buffer devoid of '~SCa2+ and filtered immediately, Filters wove washed 3 times with ti~e same buffer and counted for radioactivity in a liquid scintillation counter. Curve represented by triangles (A) shows the '~SCa2+ accumulation in the absence of ATE
20
I I00
I 2OO
i 3O0
Cd~- CONC. I~MI Fig. 2. Effect of Cd 2+ on Ca2+-ATPase activity. Ca2+-ATPase activity was measured as described in Materials and Methods. Data presentec~ are an average of 3 experiments.
129 pies are removed every 10 s. Radioactivity was measured as described in Materials and Methods. It can be seen from Fig. 3 that vanadate does not stimulate Ca2+-release in a way similar to Cd 2+ (i.e. does not show a sudden Ca 2+ release). However, a slow diffusion of Ca 2+ appeared in the presence of vanadate (possibly due to inhibition of Ca 2+ reuptake). Since vanadate does not cause a sudden release of Ca 2+ (although it definitely inhibits Ca2+-ATPase activity), this indicates that the ability of Cd 2+ to inhibit Ca2+-ATPase activity may not be responsible for the Cd2+-induced Ca 2+ release and probably involves some other mechanism. In order to investigate the mechanism of Cd2+-in duced Ca 2+ release, the effect of sulfhydryl (SH) binding agents, e.g. D T r (which was effective in case of bovine aortic smooth muscle microsomes and sarcoplasmic reticulum), is studied on Cd2+-induced effects. First the effect of these SH reagents is studied on the Ca2+-trans port. For this, 1 mM cysteine or 1 mM DTT is added to the Ca2+-loaded microsomes and the experiment is performed as described previously. It is found that cysteine or DTT alone do not have any effect on Ca 2+ transport
A
~,0
Cd~*
|
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i
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12
14
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]
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18 18 TIME (mln|
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22
(data not shown). To examine the effect of Cd 2+ in the presence of SH binding reagents, the Ca2+-loaded microsomes were preincubated with 1 mM cysteine or 1 mM dithiothreitol (DTT) prior to exposure to 100/~M C d 2+. F i g . 4 s h o w s that Cd2+-induced C a 2+ release is reduced to 47% in the presence of cysteine or DTT. In conclusion, the effect of cadmium-ions is examined on the Ca2+-transport properties in the brain microsomes. The data presented here clearly demonstrate that: (i) Cd 2+ acts as Ca2+-ATPase inhibitor and this inhibition can be reversed by DTT; and (ii) C d 2+ induces Ca2+-release which can be partially inhibited if thiol reagents were added to the microsomes prior to the Cd 2+ exposure. From our results, we suggest that Cd 2+ ions exert their effect by directly interacting with Ca 2+ATPase (to reduce the Ca2+..ATPase activity and consequently, decrease Ca 2+ uptake) in agreement with the results of Sumida et al.l~. In a previous publication by Gould et ai. 3, it has been shown that in reconstituted vesicles containing the Ca2+,Mg2+-ATPase as the only protein, the ATPase can act as a pathway for Ag+-in duced Ca 2+ efflux. Unfortunately, from our results with the microsomes, we car, not predict whether the stimulatory effect of Cd 2+ is also induced by interaction with the same protein, i.e. Ca2+-ATPase (as was suggested by Gould et al. 3 in SR) or different proteins are involved in the two processes. However, since both of these effects are reversed by thiol reagents (which maintain the sulphur-containing residues in the reduced state), these results may be attributed to oxidation of certain SH residues by Cd 2+, the reduced form/forms of which is/are essential for the enzyme (Ca2+-ATPase activity) function or Ca 2+ channel regulatory function.
!-
8O
o
X I
100 2OO 3OO 4OO Cd2+ CONCENTRATION (/AM) Fig. 3. CdZ+-induced Ca2+ release. Microsomes (I mg/ml protein)
were inr,ubated with buffer containing 100 mM KCI, 2.5 mM MgCI2, 5/~M free Ca2+, 20 mM HEPES, 0.1/~Ci 45Ca2+ and 1.6 mM ATP (pH 7.0). After 15 min incubation at 37 °C, different concentrations of C d 2+ were added to the reaction mixture and 200/~1 aliquots were removed at 10 s interval. Radioactivity was measured as described in Fig. 1. (A) Ca 2+ release at various Cd 2+ concentrations. Curve presented by broken line shows Ca2+-release by 100 /~M Na.orthovanadate. The data represented are typical of 5 experiments. (B) Dose d e p e n d e n c y of Cd 2+ -induced Ca 2+ release.
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16 18 20 TIME (min)
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Fig. 4. Effect of cysteine on Cd2+-induced Ca2+ release: Cd2+-in -
duced Ca 2+ release was measured in the presence (O) and absence (o) of 1 mM cysteine. Cd 2+ concentration was 250/~M.
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