123
Journial of Physiology (1991). 443, pp. 123-136 Wt'ith 9 figures Printed in Great Britain
CADMIUM UPTAKE THROUGH THE ANION EXCHANGER IN HUMAN RED BLOOD CELLS
BY M. LOU, R. GARAY AND J. 0. ALDA From the Departamento de Fisiologt'a, Facultad de MIedicina, 50009 Zaragoza, Spain
(Received 30 August 1990) SUMMARY
1. The initial rate of Cd2+ uptake in human red cells was measured by atomic absorption spectrophotometry. 2. About 96 % of Cd2" uptake was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid) with IC50 (concentration giving 50% of maximal inhibition) of 0 3 /JM and by furosemide with IC50 of 500 /tM and was resistant to ouabain and amiloride. This indicates the implication of the [Cl--HC03-] anion exchanger in Cd21 uptake. 3. DIDS-sensitive Cd2+ uptake required the presence of external HCO3. HC03ions had a biphasic effect on Cd21 uptake. Low bicarbonate concentrations were stimulatory, suggesting formation of translocating bicarbonate-cadmium complexes. Higher bicarbonate concentrations were inhibitory, suggesting further bicarbonate complexation with formation of non-translocating species. Depending on the presence or absence of external Cl-, a maximal Cd21 uptake of 1P7 or 0 37 mmol (1 cells)-' h-' was observed at bicarbonate concentrations of 15 6 or 11 mm respectively. 4. In the presence of bicarbonate, external Cl- ions strongly stimulated Cd2+ uptake, with linear increase between 70 and 125 mm. This suggests that one translocating species may have chloride as ligand. 5. DIDS-sensitive Cd21 uptake was modestly inhibited by physiological concentrations of external phosphate and was resistant to external K+, Mg2+ and Ca2+. 6. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HC03- such as [Cd(OH)(HCO3)2]- and
[Cd(OH)(HCO3)Clp-. INTRODUCTION
Red blood cell membranes exhibit an unusually high anion/cation flux ratio. This results from the presence of a very active membrane Cl--HCO3 anion exchanger (for review see Knauf, 1987). In consequence, residual forms of operation of this exchanger may become important transport pathways for cations in the form of anionic complexes. For instance, Na+ can reach the red cell interior through the anion exchanger in the form of NaCO3- ion pairs (Becker & Duhm, 1978; Funder, Tosteson & Wieth, 1978; Garay, Hannaert, Nazaret & Cragoe, 1986). The importance of this NaCO3- influx is illustrated by the clinical observation that patients with MS 8766
124
M. LOU, R. GARAY AND J. 0. ALDA
metabolic alkalosis, characterized by an increased HCO3- concentration in the plasma, accumulate Na+ in their erythrocytes (Funder & Wieth, 1974a, b; Funder, 1980). Recent studies have suggested that the anion carrier may play an important role in the uptake of those trace elements able to form complexes with bicarbonate, carbonate and/or chloride in significant amounts under physiological conditions: lead, probably in the form of a [PbCO3Cl]- anionic complex (Simons, 1986), zinc, probably in the form of a [Zn(HCO3)2Clf and/or [ZnOH(HCO3)Cl] anionic complex(es) (Kalfakakou & Simons, 1986, 1990; Alda & Garay, 1989) and copper, probably as [Cu(OH)2Clf- or [Cu(OH)2HC03]- (Alda & Garay, 1990). For several reasons it suggested to us that this could be the case for cadmium. First, Cl- or HC03ions can associate with Cd2+ in order to form anionic complexes (Baes & Mesmer, 1976; Phillips & Silvester, 1983; Bernard & Busnot, 1984; Prince, 1987). Second, it has been previously shown that Cd2+ is able to be transported across red cell membranes, although the mechanism was unclear (Garty, Bracken & Klaasen, 1986; Foulkes, 1988). Third, chronic cadmium toxicity is in some way associated with cadmium accumulation in red blood cells (Garty, Wong & Klaasen, 1981; Enger, Hildebrand & Stewart, 1983; Tanaka, Min, Onosaka, Fukuhara & Veda, 1985). Finally, red cell metallothionein binds cadmium (Garty et al. 1981; Enger et al. 1983; Tanaka et al. 1985). In this paper we report a study of Cd2+ transport in human red blood cells. The results suggest that the anion exchanger is the main route for the transport of Cd2+ in the form of an anionic complex with bicarbonate. METHODS
Preparation of red cells Thirty millilitres of venous blood from healthy subjects was collected in heparinized tubes and centrifuged at 1750 g for 10 min at 4 'C. The plasma and buffy coat were removed and the red cell pellet was used immediately. Measurement of ionized Cd2+ in the flux media Ionized Cd2+ concentration in the flux media was measured by using an Orion model 94-48 Cd2+selective electrode (Orion Research, Boston, MA, USA). Table 1 shows voltage readings (V) in NO3- medium as a function of the total cadmium concentrations (CdT). It can be seen that an increase in CdT from 0-1 to 10 mm induced a change in voltage reading (AV) of 29-8 + 5-3 mV. This value was similar to the AV (28-8 + 4-8 mV) obtained for a CdT change from 1-0 to 10-0 mm. Therefore, AV was a linear function of the logarithm of CdT, as expected by the negligible formation of Cd2+ complexes with NO3- in the range of CdT used in the flux media (Bernard & Busnot, 1984). Then, NO3- was used as a reference anion (see below). Table 1 shows that the preparation of cadmium solutions in Cl- media reduced the value of AV by about 1/3, indicating a modest complexation of cadmium with Cl- (for details on cadmium complexation with chloride see Bernard & Busnot, 1984). Table 2 shows the influence of bicarbonate ions on ionized cadmium concentration. It can be seen that the voltage readings were strongly diminished by increasing the HC03- concentration, indicating an important cadmium complexation. Thus, taking NO3- as reference anion, Table 2 shows that a solution containing 20 mM-bicarbonate induced a 47 % decrease in cadmium activity. Measurement of Cd2+ uptake Fresh erythrocytes were washed twice with cold 150 mM-NaCl (or 150 mM-NaNO3) and resuspended in the same solution at a haematocrit of 12-14%. Cell suspension (0 5 ml) was added to duplicates of tubes containing 2 ml of different cadmium-
CADMIUM TRANSPORT IN RED CELLS 1800
125
Control
(i 1600
- 1400 -
E 1200 Z-
1000
*
a) r
+N~0
800 600
DIDS
-,x 400c i4
c
200
A
A
A
laA
A
70 40 50 60 30 Time (min) Fig. 1. Cd2+ uptake in human red cells. It can be seen that internal Cd2+ contents increased linearly with time for at least 10-15 min. DIDS (10 /uM) inhibited an important fraction of Cd2+ influx. The initial internal Cd2+ content (at zero time) represents contaminating
10
20
Cd2+.
TABLE 1. Potentiometric reading of Cd2+ activity with a Cd2+-selective electrode in NO3- and Cl- media Voltage reading (mV) Total Cd2+ concentration Cl-(150 mM) (mM) NO3-(150 mM) 178-6+ 3-5 175-2+4-1 0.1 152-6+3-1 145-4+3-1 1.0 132-8+2-9 -116-6+3-6 10-0 Values are means+ S.D. of six experiments. NO3- medium contained 150 mM-NaNO3 and different Cd(NO3)2 concentrations. Cl- medium contained 150 mM-NaCl and different CdCl2 concentrations. -
-
-
-
TABLE 2. Potentiometric reading of Cd2+ activity with a Cd2+-selective electrode: influence of the HCO3- composition Cadmium activity Voltage reading HCO3- concentration (mM) (mV) (mM) 0 0-1 198-7+4-5 0-091 199-7+4-3 5 -201-0+4-1 0-082 10 0-053 -206-6+3-9 20 0-034 30 -212-3+4-3 -223-9+4-2 0-014 40 anion. Bicarbonate reference was used as three NO3Values are means ± S.D. of experiments. isosmotically replaced NO3,- The final solutions contained 0-1 mM-Cd(NO3)2 and final pH was adjusted to 7-4 (at 37 °C) with MOPS-Tris buffer. -
-
containing media. A basic Cd2+-medium contained (mM): CdCl2, 140 NaCl, 5 KCl, 1 MgC12, 1 CaCl2, 10 glucose and 10 MOPS(3-[N-morpholino]propanesulphonic acid)-Tris (pH 7-4 at 37 °C), where x varied from 10 to 1000,C6M. In some experiments Cl- was replaced isosmotically by NO8-, HCO-, HP042-/H2PO4- and SO42-/HSO04 NO3- can move through the Cl--HCO3- exchanger and, x
126
M. LOUL R. GARA Y AND J. 0. ALDA
in the range of ion concentrations used in the study, cadmium complexes with nitrate were negligible (see above). Therefore NO3- was used as a reference anion. In other experiments, Na' was replaced isosmotically by K+, Mg2+ or Ca2+. All flux media were freshly prepared every day and the osmolarity was kept constant at 295+5 mosM 1'. The tubes were capped and incubated at 37 °C for 0. 5, 10, 20 and 30 min. In control experiments we verified that external pH remained almost constant (±004 pH units) during the incubation period. To stop the reaction. tubes were chilled at 4 °( for 1 min and then centrifuged for 3 min at 1750 g at 4 'C. The supernatants were removed an(l the red cell pellet washed three times with 150 mm-NaNO3 or 150 mM-NaCl. At the end of the last wash, the red cell pellet was haemolyzed with 4 ml of 0-02 % Acationox (Monoject Scientific, St Louis, MO, USA). The tubes were centrifuged for 20 min at 5000 g. The supernatants were transferred into tubes for Cd2" analysis in an atomic absorption spectrophotometer (Carl Zeiss M4QIII, Oberkochen, Germany) and for measurement of haemoglobin absorbance at 541 nm. Cd2+ standards were prepared by dilution in Acationox, 002%. In control experiments we verified that the Cd2' readings did not undergo interference by the haemolysates (similar Cd2' readings were obtained by adding Cd2+ to distilled water or to Acationox-treated haemolysates). Figure 1 shows internal Cd2+ content as a function of time. The initial rate of Cd2+ uptake was calculated from the initial slope of this function. In all flux media. cell Cd2+ content increased linearly with time for at least 10-15 min. Therefore. fluxes were measured by using incubation times of 7-5-10 min. Figure 1 shows that DIDS was able to inhibit an important fraction of CId2+ influx. DIDS-sensitive Cd2+ uptake was taken as a marker of Cd2+ influx through the anion exchanger (for details on DIDS-sensitive cation fluxes see Becker & Duhm. 1978; Funder et al. 1978; Garay et al. 1986).
Measurement of HCO3- concentration in the flux media Capillary tubes were filled with aliquots of flux media, and were then sealed at both ends as a standard. Another set of capillaries was filled with the supernatants of the cell suspensions, taken at the end of the flux experiment. The capillaries were equilibrated for 10 min and the HCO3- concentrations were measured in an AVL-945 gas analyser (Instrumental Biomed, Graz, Austria). In control experiments we verified that values of HCO3- concentrations were stable (within +05%) for equilibration times between 0 and 30 min. In all cases, the pH of the flux media was measured by using a Ross electrode (Orion Research, Boston, MIA, USA). The measurements of HC03- concentrations and pH. before and after incubation with the cells, exhibited in almost all cases a variation less than 3%,. The mean values were used for kinetic analysis or represented in the figures. The effect of drugs To study the effect of drugs on red cell Cd2+ uptake, concentrated stock solutions of the compounds were prepared by dilution in water. ethanol or dimethyl sulphoxide. The cell suspensions (in C1--HCO3- media, Cd2+-free, containing C1- 130 mm, HC03- 15 mM) were preincubated for 3 min (at 4 °C) with different concentrations of compounds (final concentrations of ethanol and dimethyl sulphoxide did not modify ion transport per se). Finally, CdCl2 was added up to a final concentration of 100 /IM. Furosemide was obtained from Hoechst Laboratory (La Defense, Paris. France). Amiloride was a gift from Merck, Sharp & Dohme Laboratories (Paris. France). Bumetanide was obtained from Leo Laboratory (Vernouillet. France). DIDS, SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'disulphonic acid) and all other chemicals were either from Merck or Sigma (distributed through
Coger. Paris, France). RESULTS
Pharmacological properties of Cd2+ uptake in human red blood cells Several ion transport inhibitors were tested on the initial rate of red cell Cd2+ uptake (in bicarbonate and chloride medium containing CdCl2, 100 UtM). Figure 2 shows that DIDS inhibited about 95 % of Cd2+ uptake with an IC50 (concentration
CADMIUM TRANSPORT IN RED CELLS
127
100 0
80
-
0
0
o
60 0
X 40. 20
0
0
0-1
1 10 DIDS concentration (,M)
100
Fig. 2. Inhibition of red cell Cd2+ uptake by DIDS. It can be seen that DIDS was able to inhibit about 95 % of Cd2+ uptake with ICO of 0 3 /Mm. Fluxes were measured in Cl--HCO3medium containing 130 mm- Cl-, 15 mm- HCO3- and 100 ,tM-CdCl2. The same results were obtained in three other experiments. -C ., r-
0
2
E E
a, 0 +Cu
0)
ax
0 0-25 0-5 0.7 External Cd2+ concentration (mM)
-1.0 -0.75 -0.5 -0.25
Cl) C]
1-2 0.8 1-0 0.6 0-4 External Cd2+ concentration (mM) Fig. 3. DIDS-sensitive Cd2+ uptake as a function of external Cd2+ concentrations ([Cd2].). Cd2+ uptake reached saturation at high [Cd2+]0. Cd2+ uptake was measured in bicarbonate (2 mM) and chloride medium. Values represent means of triplicates (the range of variation was smaller than the size of the symbols). Inset, Hanes plot of the data. Apparent dissociation constant for [Cd2+]. (Kcd) was obtained from the intercept with the x-axis and Vmax Cd, the maximal rate of Cd2+ uptake, was obtained from the slope. KCd was about 1 mm and Vmax Cd about 4-3 mmol (1 cells)-' h-'. 0-0
0.2
giving 50 % of maximal inhibition) of 0-3 /tM. A similar extent of inhibition was obtained with SITS (IC50 = 0-3 /tM), xipamide (IC50 = 80,M), phloretin (IC50 = 100 /IM), and furosemide (IC50 = 500 LlM). Partial inhibition was obtained with high concentrations of bumetanide (700 ,UM) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) (120 /kM). Ouabain and amiloride were without inhibitory effect on Cd2+ uptake. This pharmacological pattern agrees well with that of anion carrier
-|
M. LOU, R. GARAY AND J. 0. ALDA
128 A r-
400
-c
_ 350* C.) _ 300 .5 E Zs. 250
0
/~~~~~~~~ 0
0
X 200
0
0
a
+A 150 a) 100 0)
50.
Cl c]
O
C
en 5
15 20 25 30 External HC03- (mM)
10
B
30
25
.i 0.
20
+
15
-
0
10
-6
-4
-2
4
6 External HCO3- (mM) 02
35
8
10
0
40
45
12
Fig. 4. A, DIDS-sensitive Cd2+ uptake as a function of external bicarbonate concentrations ([HCO3-]0) in nitrate medium. It can be seen that Cd24 uptake was: (i) stimulated by low [HCO3-]0 up to a maximal Cd2+ uptake of about 400 4umol (1 cells)-' h-' at [HCO3-]0 t 11 mm and (ii) inhibited by excess [HCO3-]j. Bicarbonate was replaced isosmotically by
nitrate. Similar results were obtained in three other experiments. B, Hanes plot of the first, stimulatory phase (bicarbonate concentrations from 0 to 10 mM). Apparent dissociation constant for [HCO3-0 (KbiC) was obtained from the intercept with the x-axis and Vmax,bic, the maximal rate of Cd2+ uptake, was obtained from the slope. Linearity was obtained with n = 1. In four different experiments we obtained KbiC = 5-7 + 0-4 mm and
Vmax,bic = 710±19,umol (1 cells)-' h-' (mean +S.D.). fluxes (Garay et al. 1986). Therefore, we equated Cd2+ fluxes catalysed by the anion carrier to those inhibited by 10 /IM of DIDS. Stimulation of Cd2+ uptake by external Cd2+
DIDS-sensitive Cd2+ uptake
was
measured in human red cells incubated in ([Cd2+]O).
bicarbonate and chloride medium containing different Cd2+ concentrations
CADMIUM TRANSPORT IN RED CELLS
129
A
1800 T 1600 1400 0
1200
-o
1000
0
/~~~0\ / \
X800 +
600
/ -
*> 400- O C)I 200 /
0
5
10
30 20 25 15 External HCO3- (mM)
35
40
45
15
20
10.
B 0. +
0
-20
-15
-10
-5
8-
66-
0
5
10
External HC03- (mM)
Fig. 5. A, DIDS-sensitive Cd'+ uptake as a function of external HCO3- concentrations ([HCO,-]0) in chloride medium (105 mM). Cd2+ uptake was qualitatively similar and higher than in chloride-free medium (Fig. 4A). Moreover, maximal Cd2+ uptake was found at [HCO.3-]o 15 mm. External HCO - was isosmotically replaced by NO3-. External Cd2 concentration was 100 /M. Similar results were obtained in four other experiments. B Hanes plot of the first, stimulatory phase (bicarbonate concentrations from 0 to 15 mM). 189+1-2mM and Vm..,ic= In five different experiments we obtained K -i,= 3860 +430 ,tmol (1 cells)-' h-l (mean + S.D.).
Figure 3 shows that in the 10-200 fM [Cd2+] range, Cd2+ uptake was almost a linear function of [Cd2+]O, and that high external Cd2+ concentrations were required to reach saturation. Indeed, a Hanes plot of the data (Fig. 3, inset) revealed an apparent dissociation constant for external Cd2+ (KCd) of about 1 mm and a maximal 5
PHY 443
M. LOU, R. GARAY AN,D J. 0. ALDA
130
rate (Vmax) of about 4 3 mmol (1 cells)-1 h-1 (for details of Hanes plots see Garay & Garrahan, 1973). In all following experiments we used flux media containing 100 ,am-Cd2".
Stimulation of Cd2+ uptake by external bicarbonate In studying the effect of bicarbonate on Cd21 uptake, we measured initial and final [HCO3j0 as described in Methods. Experiments where [HC03-j varied more than 5 % were rejected. s 2.0 n 1.8
a.)/
- 1.6
E 1 .4 E
1.2.
0.8/
+
~0*4 0~~~~~~~~~~~~~~ C_
$n o
0-
0
20
40 80 100 120 60 External Cl- concentration (mM)
140
160
Fig. 6. DIDS-sensitive Cd2+ uptake as a function of external chloride concentration ([Cl-]l). It can be seen that: (i) in the presence of 15 mM-bicarbonate (A), Cd2" uptake was strongly stimulated by increasing [Cl-]o following a non-saturable function and (ii) in media without added bicarbonate (0), [Cl-]0 had almost no effect on Cd2" uptake. External Cl- was isosmotically replaced by different N03- concentrations. External Cd2+ concentration was 100 /iM.
In chloride- and bicarbonate-free media (NO3- medium), DIDS-sensitive Cd2+ uptake was very small (less than 0 05 mmol (1 cells)-' h-'; contaminating bicarbonate from atmospheric CO2 was about 0 5 mm). Adding bicarbonate strongly stimulated Cd21 uptake, following a complex function. Figure 4A shows that the stimulation of Cd2+ uptake exhibited inhibition by excess [HCO3-j0, with a maximal Cd2+ uptake of about 400 /tmol (1 cells)-1 h-' at a bicarbonate concentration of about 11 mm. Figure 4B shows a Hanes plot of the first, stimulatory phase (between 0 and 10 mm [HCO3-j0). In four different experiments we obtained an apparent dissociation constant for [HCO3-j0 (KbiC) of 5*7 +0 4 mm (mean + S.D.) and Vmax = 710+ 19 ,tmol (1 cells)-' h-1. A more important stimulation by bicarbonate was obtained in chloride media. Figure 5A shows a maximal Cd2+ uptake of about 1700 jtmol (1 cells)-' h-1 at a bicarbonate concentration of about 16 mm. Figure 5B shows a Hanes plot of the first, stimulatory phase (between 0 and 15 mm [HCO3-j0). In five different
CADMIUM TRANSPORT IN RED CELLS
131
experiments we obtained an apparent dissociation constant for [HCO3-j0 (KbiC) of 18-9 + 1-2 mm (mean+ S.D.) and Vmax = 3860 + 430 /tmol (1 cells)-' h-1.
Stimulation of Cd2+ uptake by external chloride Figure 6 shows that: (i) in media without added bicarbonate, chloride ([Cl-]0) had almost no effect on DIDS-sensitive Cd2+ uptake (at [Cl-] = 145 mm, DIDS-sensitive , 2500 0
2250 @ 2000 o 1750
E
0
1500
1250 + 1000
, 750 ,, 500
c,
p/ s
250
o
6.0
6.2
6.4
6.6
6.8 7-0 7.2 External pH
7.4
7.6
7-8
8.0
Fig. 7. Stimulation of DIDS-sensitive Cd2+ uptake by alkalinization. Circles and squares indicate two different experiments. It can be seen that a change in pH from 6-2 to 7-8 stimulated Cd2+ uptake by more than one order of magnitude. The flux medium contained 130 mm of chloride and 15 mm of bicarbonate. External pH was changed by addition of MES, 2-(N-morpholino)ethanesulphonic acid (6-6 8), MOPS and Tris. External Cd2+ concentration was 100 fM.
Cd2+ uptake was lower than 80 jtmol (1 cells)-' h-1) and (ii) in the presence of bicarbonate, Cd2+ uptake was strongly stimulated by [Cl-]0. This stimulatory function did not reach saturation at physiological chloride concentrations (Fig. 6). Our data allowed us to estimate that under physiological chloride and bicarbonate concentrations and a cadmium concentration of 100 saM, DIDS-sensitive Cd2+ uptake should be ~ 2000 ,umol (1 cells)-' h-1. Stimulation of Cd2+ uptake by alkalinization DIDS-sensitive Cd2+ uptake exhibited profound stimulation by alkalinization. Figure 7 shows that a change in pH from 6-2 to 7-8 increases Cd2+ uptake by more than one order of magnitude. In addition, the data suggest that the inflexion of the stimulatory function was close to physiological values of pH (Fig. 7). Inhibition of Cd21 uptake by phosphate Figure 8A shows DIDS-sensitive Cd2+ uptake as a function of external phosphate, in media containing different bicarbonate concentrations. It can be seen that phosphate was a modest inhibitor of Cd21 uptake. In bicarbonate media, high 5-2
M. LOU, R. GARAY AND J. 0. ALDA
132
phosphate concentrations were required to inhibit Cd2+ uptake. Indeed, no more than 25% inhibition was observed at physiological external phosphate concentrations (Fig. 8A). Figure 8B shows a Dixon plot of the data. In five different experiments (in bicarbonate media) we obtained a Kpo4 (apparent dissociation constant for external phosphate) of 35 +04 mm (mean ±S.D.). A
2.0 4) a) 0.
1.5 U-
a)en
C.)
+U1)
a)U) E C,)
10. 1.0
I.5
E
A
a a
A
*M--A~~~~
A
ol - ^-
0.0
10
5
0
-
.0
20
15
External phosphate (mM)
B
0-061 a)
0.05
C0. 0
0-04
0
C
j /~~U I~~~
0-03
0
._
.0 0.02 .
C
0-01.6
0~~~~~
A__... A, "o
-5
, -.W W
0
Ah,
0-
5
10
15
20
External phosphate (mM)
Fig. 8. A, inhibition of DIDS-sensitive Cd2+ uptake by external phosphate. Flux media contained different bicarbonate concentrations (Z = without added HCO3-, A = 7-5 mM-HCO3- and * = 15 mM-HCO3). It can be seen that high phosphate concentrations were required to inhibit Cd21 uptake. Flux media contained chloride as major monovalent anion. External Cd2+ concentration was 100 sm. Similar results were obtained in four other experiments. B, Dixon plot of the inhibition of Cd2+ uptake by phosphate (data from Fig. 8A). In five different experiments (in bicarbonate media) we obtained a KP04 (apparent dissociation constant for external phosphate) of 3.5 + 04 mM (mean +S.D.).
CADMIUM TRANSPORT IN RED CELLS
133
Other ions Sulphate was a very modest inhibitor of DIDS-sensitive Cd2+ uptake (it inhibited about 10 % of Cd2+ uptake at a concentration of 20 mM). Physiological concentrations of cations Na+, Mg2+ and Ca2+ were unable to modify Cd2+ uptake. In addition, Cd2+ 1200
a) 1000
A7
E 800
~~~~~~~~~A/00O
x
600 cL 600
^A
40. +
400
7A
,200
7.-g
DoCh 0
5
10 15 20 25 30 35 40 External HC03- concentration (mM)
45
Fig. 9. Stimulation of DIDS-resistant red cell Cd2+ uptake by external bicarbonate. It can be seen that bicarbonate had more stimulatory action in nitrate (A) than in chloride (D) media.
uptake was insensitive to external K+ (isosmotic replacement of Na+ by K+ was unable to modify Cd2+ uptake).
Superoxide dismutase The anion carrier is able to transport superoxide anions. However, superoxide dismutase (SOD, 500 U ml-') was unable to modify DIDS-sensitive Cd2+ uptake.
DIDS-resistant red cell Cd2+ uptake DIDS-resistant Cd2+ uptake was resistant to ouabain, amiloride and phloretin and increased linearly with external Cd2+ concentration (data not shown). In addition, Fig. 9 shows that it was stimulated by [HCO3-j0 in nitrate media (a less important stimulation was observed in chloride media). Therefore, DIDS-resistant Cd2+ uptake in nitrate-bicarbonate media was higher than in chloride-bicarbonate media (about 50% vs. 5% in Fig. 2). Red cell Cd2+ efflux In contrast with Cd2+ uptake, the initial rate of Cd2+ efflux was undetectable and was independent of the presence or absence of external Ca2 . Indeed, in erythrocytes loaded up to 1 mmol (1 cells)-' of total cadmium content, the initial rate of Cd2+ efflux, if any, was less than 1 ,umol (1 cells)-' h-1.
134
M. LOU, R. GARAY AND J. 0. ALDA DISCUSSION
In bicarbonate-containing medium, red blood cells exhibit high Cd21 influx. For instance, Cd2+ fluxes in Figs 4 and 5 are similar in magnitude to the Na+ and K+ fluxes catalysed by the red cell Na+-K+ pump. This and the high sensitivity of the atomic absorption methods for cadmium (with standard error lower than 1-2 %) allowed us to precisely measure cadmium movements across human red cell membranes. Our values for Cd2+ uptake are higher than those previously obtained by Garty et al. (1986) in rat red blood cells. This may result from: (i) a difference between rat and human red blood cells and/or (ii) the fact that Garty et al. worked in Cl- medium without added HC03- Moreover, in contrast with 'Garty et al. (1986), we were unable to detect an initial rapid phase of Cd2+ uptake (Cd2+ influx was linear for more than 7 min). This can be explained by the fact that, unlike Garty et al. (1986), we have discarded the cell membranes. Therefore, the rapid phase of Cd2+ uptake may represent Cd2+ adsorption to the cell membrane. One technical problem in measuring red cell Cd2+ fluxes is to maintain constant medium HC03- concentrations. In media without HC03- added some is formed from atmospheric and metabolic (red cell-generated) CO2. In bicarbonate-containing media, some HC03- can be lost through CO2 evaporation. In order to avoid potential artifacts we capped the tubes during the flux experiment, we controlled the external pH and we measured systematically the initial and final external bicarbonate concentrations. Changes in external bicarbonate concentrations during the flux experiment were lower than 5-10 % of initial values. A screening of transport inhibitors showed that DIDS and furosemide inhibited a very important fraction of red cells Cd2+ uptake with IC50 similar to those classically obtained for anion carrier fluxes (for references see Gunn, 1979; Garay et al. 1986). Indeed, at physiological bicarbonate concentrations, DIDS-sensitive Cd2+ uptake was about 95 % of the total Cd2+ uptake. This is in line with Garty et al. (1986) who found that Cd2+ uptake in rat red blood cells was resistant to metabolic inhibitors, suggesting passive transport. Catalysis of Cd2+ uptake by the anion exchanger requires Cd2+ to be transported in the form of an anionic complex, particularly a monovalent anion. On the other hand, Cd2+ uptake was strongly dependent on the presence of bicarbonate. This suggests that the translocating anionic species has at least one bicarbonate ion. In addition, it can have one OH- ion because the pKD (-log of dissociation constant) for the Cd-OH+ ion pair is about 7-6 (Bernard & Busnot, 1984). Finally, chloride strongly stimulated bicarbonate-dependence Cd2+ uptake. Taken together these findings suggest that one translocating species can be the monovalent anion
complex:
[Cd(OH)HCO3Cl]-.
On the other hand, a significant, bicarbonate-dependent Cd2+ uptake was observed in N03- medium. Therefore, another translocating species can be the [Cd(OH)(HCO3)2] monovalent anion complex. However, we cannot exclude the possibility that other minor anionic cadmium complexes (Baes & Mesmer, 1976; Bernard & Busnot, 1984; Prince, 1987) such as
CADMIUM TRANSPORT IN RED CELLS 135 [Cd(HCO3)2Cl]-, [Cd(OH)CO3]-, [Cd(CO3)Cl]- and [Cd(OH)(Cl)2 can also be transported by the anion carrier. The red cell anion exchanger is able to catalyse much higher fluxes for monovalent than for divalent anions (Gunn, 1979). Therefore, the inhibition of Cd2+ uptake by excess bicarbonate may be interpreted as resulting from additional bicarbonate complexation, with the formation of divalent anionic species such as [Cd(OH)-
(HCO3)3]2-, [Cd(CO3)2]2- or [Cd(OH)(HCO3)2C1]2-. Regarding the pH dependence, DIDS-sensitive Cd2+ uptake exhibited the opposite tendency to that previously reported for anion carrier fluxes (Gunn, 1979), i.e. it was stimulated by alkalinization (from pH 7-0 to 7 8). This can be explained by an increase in the concentration of the translocating species [Cd(OH)HCO3Cl]- and [Cd(OH)2HCO3)]-, due to the increase in OH- concentration. Besides bicarbonate and chloride, phosphate was a physiological anion able to modify cadmium uptake. Modest inhibition of cadmium uptake by physiological concentrations of phosphate may be explained by formation of a non-translocating species such as Cd3(PO4)2. Red cell Cd2+ uptake may have important toxicological consequences. Thus, cadmium exposure from dietary sources (30-50 jtg day-'; Yost, 1984) results in continuous absorption from ingested food into blood where it is transported and delivered to target tissues. Blood cadmium reaction can be categorized into two compartments: (i) a rapid one, clearing about 99 % of acutely administered cadmium within 3 h and (ii) a slow one in red blood cells, accounting for chronic cadmium exposure (Garty et al. 1981). Our results suggest that the slow exchanges between red cells and plasma are mediated by the anion carrier. However, it is important to note that: (i) red cell Cd2+ uptake in vivo is strongly limited by the extent of cadmium bound to proteins and (ii) rate constants of outward Cd2+ movements were undetectable, suggesting that haemoglobin and/or other intracellular proteins bind Cd2+ with high affinity and high capacity. It appears therefore that determination of in vivo Cd2+ uptake requires further investigation. Besides erythrocytes, the anion carrier appears to exist in the kidney, where it may participate in bicarbonate reabsorption (Schild, Giebisch, Karniski & Aronson, 1986). Whether the anion carrier is involved in the renal handling of cadmium deserves further investigation. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HCO3- such as [Cd(OH)(HCO3)2]- and [Cd(OH)(HCO3)Cl]-. This study was supported by the Ion Transport and Hypertension Association, France. REFERENCES
ALDA, J. 0. & GARAY, R. (1989). Evidence for a major route for zinc uptake in human red blood cells: [Zn(HCO3)2C1]- influx through the [Cl-/HC03-] anion exchanger. Journal of Cellular Physiology 138, 316-323. ALDA, J. 0. & GARAY, R. (1990). Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells. American Journal of Physiology 259, C570-576.
M. LOU, R. GARAY AND J. 0. ALDA BAES, C. F. & MESMER, R. E. (1976). In The Hydrolysis of Cations, pp. 265-274. John Wiley & Sons,
136
New York. BECKER, B. F. & DUHM, J. (1978). Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions. Journal of Physiology 282, 149-168. BERNARD, M. & BuSNOT, F. (1984). In Usuel de Chimie Gene'rale et Minerale: Solutions Aqueuses, section D, Complexes, pp. 230-248. Bordas, Paris, France. ENGER, K.D., HILDEBRAND, C. D. & STEWART, C. D. (1983). Cd responses of cultured human blood cells. Toxicology and Applied Pharmacology 69, 214-224. FOULKES, E. C. (1988). On the mechanism of transfer of heavy metals across cell membranes. Toxicology 52, 263-272. FUNDER, J. (1980). Alkali metal cation transport through the human erythrocyte membrane by the anion exchange mechanism. Acta Physiologica Scandinavica 108, 31-37. FUNDER, J., TOSTESON, D. C. & WIETH, 0. (1978). Effects of bicarbonate on lithium transport in human red cells. Journal of General Physiology 72, 233-247. FUNDER, J. & WIETH, 0. (1974a). Human red cell sodium and potassium in metabolic alkalosis. Scandinavian Journal of Clinical and Laboratory Investigation 34, 49-59. FUNDER, J. & WIETH, 0. (1974b). Combined effects of digitalis therapy and of plasma bicarbonate on human red cell sodium and potassium. Scandinavian Journal of Clinical and Laboratory Investigation 34, 153-160. GARAY, R. P. & GARRAHAN, P. J. (1973). The interaction of sodium and potassium with the sodium pump in red cells. Journal of Physiology 231, 297-325. GARAY, R. P., HANNAERT, P. A., NAZARET, C. & CRAGOE, E. J. JR (1986). The significance of the relative effects of loop diuretics and anti-brain edema agents on the Na+, K+, Cl- cotransport system and the Cl-/NaCO3- anion exchanger. Naunyn Schmiedeberg's Archives of Pharmacology 334, 202-209. GARTY, M., BRACKEN, W. M. & KLAASEN, C. D. (1986). Cadmium uptake by rat red blood cells. Toxicology 42, 111-119. GARTY, M., WONG, K. L. & KLAASEN, C. D. (1981). Redistribution of cadmium to blood of rats. Toxicology and Applied Pharmacology 59, 548-554. GUNN, R. B. (1979). Transport of anions across red cell membranes. In Membrane Transport in Biology, vol. II, Transport across single biological membranes, ed. GIEBISCH, G., TOSTESON, D. C. & USSING, H. H. pp. 59-80. Springer-Verlag, Berlin. JENNINGS, M. L. (1985). Kinetics and mechanism of anion transport in red blood cells. Annual Review of Physiology 47, 519-533. KALFAKAKOU, V. & SIMoNs, T. J. B. (1986). The mechanism of zinc uptake into human red blood cells. Journal of Physiology 381, 75P. KALFAKAKOU, V. & SIMoNs, T. J. B. (1990). Anionic mechanisms of zinc uptake across the human red cell membrane. Journal of Physiology 421, 485-498. KNAUF, P. A. (1987). Anion transport in erythrocytes. In Membrane Physiology, ed. ANDREOLI, T.E., HOFFMAN, J. F., FANESTIL, D. D. & SCHULTZ, S. G., pp. 191-220. Plenum Press, New York. PHILLIPS, S. L. & SILVESTER, L. F. (1983). Use of balanced-like-charges approach to metal-bicarbonate reactions. Inorganic Chemistry 22, 3848-3851. PRINCE, R. H. (1987). Zinc and cadmium. In Comprehensive Coordination Chemistry, vol. V, ed. WILKINSON, G., GILLARD, R. D. & MCCLEVERTY. J. A. pp. 926-1004, Pergamon Press, Oxford. SCHILD, L., GIEBISCH, L., KARNISKI, L. & ARONSON, P. S. (1986). Chloride transport in the mammalian proximal tubule. Pfluigers Archiv 407, suppl. 2, S156-159. SIMONS, T. J. B. (1986). The role of anion transport in the passive movement of lead across the human red cell membrane. Journal of Physiology 378, 287-312. TANAKA, K., MIN, S., ONOSAKA, C., FUKUHARA, C. & UEDA, M. (1985). The origin of metallothionein in red blood cells. Toxicology and Applied Pharmacology 78, 63-68. YOST, K. J. (1984). Cadmium, the environment and human health: an overview. Experientia 40, 157-168.
Journial of Physiology (1991). 443, pp. 123-136 Wt'ith 9 figures Printed in Great Britain
CADMIUM UPTAKE THROUGH THE ANION EXCHANGER IN HUMAN RED BLOOD CELLS
BY M. LOU, R. GARAY AND J. 0. ALDA From the Departamento de Fisiologt'a, Facultad de MIedicina, 50009 Zaragoza, Spain
(Received 30 August 1990) SUMMARY
1. The initial rate of Cd2+ uptake in human red cells was measured by atomic absorption spectrophotometry. 2. About 96 % of Cd2" uptake was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid) with IC50 (concentration giving 50% of maximal inhibition) of 0 3 /JM and by furosemide with IC50 of 500 /tM and was resistant to ouabain and amiloride. This indicates the implication of the [Cl--HC03-] anion exchanger in Cd21 uptake. 3. DIDS-sensitive Cd2+ uptake required the presence of external HCO3. HC03ions had a biphasic effect on Cd21 uptake. Low bicarbonate concentrations were stimulatory, suggesting formation of translocating bicarbonate-cadmium complexes. Higher bicarbonate concentrations were inhibitory, suggesting further bicarbonate complexation with formation of non-translocating species. Depending on the presence or absence of external Cl-, a maximal Cd21 uptake of 1P7 or 0 37 mmol (1 cells)-' h-' was observed at bicarbonate concentrations of 15 6 or 11 mm respectively. 4. In the presence of bicarbonate, external Cl- ions strongly stimulated Cd2+ uptake, with linear increase between 70 and 125 mm. This suggests that one translocating species may have chloride as ligand. 5. DIDS-sensitive Cd21 uptake was modestly inhibited by physiological concentrations of external phosphate and was resistant to external K+, Mg2+ and Ca2+. 6. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HC03- such as [Cd(OH)(HCO3)2]- and
[Cd(OH)(HCO3)Clp-. INTRODUCTION
Red blood cell membranes exhibit an unusually high anion/cation flux ratio. This results from the presence of a very active membrane Cl--HCO3 anion exchanger (for review see Knauf, 1987). In consequence, residual forms of operation of this exchanger may become important transport pathways for cations in the form of anionic complexes. For instance, Na+ can reach the red cell interior through the anion exchanger in the form of NaCO3- ion pairs (Becker & Duhm, 1978; Funder, Tosteson & Wieth, 1978; Garay, Hannaert, Nazaret & Cragoe, 1986). The importance of this NaCO3- influx is illustrated by the clinical observation that patients with MS 8766
124
M. LOU, R. GARAY AND J. 0. ALDA
metabolic alkalosis, characterized by an increased HCO3- concentration in the plasma, accumulate Na+ in their erythrocytes (Funder & Wieth, 1974a, b; Funder, 1980). Recent studies have suggested that the anion carrier may play an important role in the uptake of those trace elements able to form complexes with bicarbonate, carbonate and/or chloride in significant amounts under physiological conditions: lead, probably in the form of a [PbCO3Cl]- anionic complex (Simons, 1986), zinc, probably in the form of a [Zn(HCO3)2Clf and/or [ZnOH(HCO3)Cl] anionic complex(es) (Kalfakakou & Simons, 1986, 1990; Alda & Garay, 1989) and copper, probably as [Cu(OH)2Clf- or [Cu(OH)2HC03]- (Alda & Garay, 1990). For several reasons it suggested to us that this could be the case for cadmium. First, Cl- or HC03ions can associate with Cd2+ in order to form anionic complexes (Baes & Mesmer, 1976; Phillips & Silvester, 1983; Bernard & Busnot, 1984; Prince, 1987). Second, it has been previously shown that Cd2+ is able to be transported across red cell membranes, although the mechanism was unclear (Garty, Bracken & Klaasen, 1986; Foulkes, 1988). Third, chronic cadmium toxicity is in some way associated with cadmium accumulation in red blood cells (Garty, Wong & Klaasen, 1981; Enger, Hildebrand & Stewart, 1983; Tanaka, Min, Onosaka, Fukuhara & Veda, 1985). Finally, red cell metallothionein binds cadmium (Garty et al. 1981; Enger et al. 1983; Tanaka et al. 1985). In this paper we report a study of Cd2+ transport in human red blood cells. The results suggest that the anion exchanger is the main route for the transport of Cd2+ in the form of an anionic complex with bicarbonate. METHODS
Preparation of red cells Thirty millilitres of venous blood from healthy subjects was collected in heparinized tubes and centrifuged at 1750 g for 10 min at 4 'C. The plasma and buffy coat were removed and the red cell pellet was used immediately. Measurement of ionized Cd2+ in the flux media Ionized Cd2+ concentration in the flux media was measured by using an Orion model 94-48 Cd2+selective electrode (Orion Research, Boston, MA, USA). Table 1 shows voltage readings (V) in NO3- medium as a function of the total cadmium concentrations (CdT). It can be seen that an increase in CdT from 0-1 to 10 mm induced a change in voltage reading (AV) of 29-8 + 5-3 mV. This value was similar to the AV (28-8 + 4-8 mV) obtained for a CdT change from 1-0 to 10-0 mm. Therefore, AV was a linear function of the logarithm of CdT, as expected by the negligible formation of Cd2+ complexes with NO3- in the range of CdT used in the flux media (Bernard & Busnot, 1984). Then, NO3- was used as a reference anion (see below). Table 1 shows that the preparation of cadmium solutions in Cl- media reduced the value of AV by about 1/3, indicating a modest complexation of cadmium with Cl- (for details on cadmium complexation with chloride see Bernard & Busnot, 1984). Table 2 shows the influence of bicarbonate ions on ionized cadmium concentration. It can be seen that the voltage readings were strongly diminished by increasing the HC03- concentration, indicating an important cadmium complexation. Thus, taking NO3- as reference anion, Table 2 shows that a solution containing 20 mM-bicarbonate induced a 47 % decrease in cadmium activity. Measurement of Cd2+ uptake Fresh erythrocytes were washed twice with cold 150 mM-NaCl (or 150 mM-NaNO3) and resuspended in the same solution at a haematocrit of 12-14%. Cell suspension (0 5 ml) was added to duplicates of tubes containing 2 ml of different cadmium-
CADMIUM TRANSPORT IN RED CELLS 1800
125
Control
(i 1600
- 1400 -
E 1200 Z-
1000
*
a) r
+N~0
800 600
DIDS
-,x 400c i4
c
200
A
A
A
laA
A
70 40 50 60 30 Time (min) Fig. 1. Cd2+ uptake in human red cells. It can be seen that internal Cd2+ contents increased linearly with time for at least 10-15 min. DIDS (10 /uM) inhibited an important fraction of Cd2+ influx. The initial internal Cd2+ content (at zero time) represents contaminating
10
20
Cd2+.
TABLE 1. Potentiometric reading of Cd2+ activity with a Cd2+-selective electrode in NO3- and Cl- media Voltage reading (mV) Total Cd2+ concentration Cl-(150 mM) (mM) NO3-(150 mM) 178-6+ 3-5 175-2+4-1 0.1 152-6+3-1 145-4+3-1 1.0 132-8+2-9 -116-6+3-6 10-0 Values are means+ S.D. of six experiments. NO3- medium contained 150 mM-NaNO3 and different Cd(NO3)2 concentrations. Cl- medium contained 150 mM-NaCl and different CdCl2 concentrations. -
-
-
-
TABLE 2. Potentiometric reading of Cd2+ activity with a Cd2+-selective electrode: influence of the HCO3- composition Cadmium activity Voltage reading HCO3- concentration (mM) (mV) (mM) 0 0-1 198-7+4-5 0-091 199-7+4-3 5 -201-0+4-1 0-082 10 0-053 -206-6+3-9 20 0-034 30 -212-3+4-3 -223-9+4-2 0-014 40 anion. Bicarbonate reference was used as three NO3Values are means ± S.D. of experiments. isosmotically replaced NO3,- The final solutions contained 0-1 mM-Cd(NO3)2 and final pH was adjusted to 7-4 (at 37 °C) with MOPS-Tris buffer. -
-
containing media. A basic Cd2+-medium contained (mM): CdCl2, 140 NaCl, 5 KCl, 1 MgC12, 1 CaCl2, 10 glucose and 10 MOPS(3-[N-morpholino]propanesulphonic acid)-Tris (pH 7-4 at 37 °C), where x varied from 10 to 1000,C6M. In some experiments Cl- was replaced isosmotically by NO8-, HCO-, HP042-/H2PO4- and SO42-/HSO04 NO3- can move through the Cl--HCO3- exchanger and, x
126
M. LOUL R. GARA Y AND J. 0. ALDA
in the range of ion concentrations used in the study, cadmium complexes with nitrate were negligible (see above). Therefore NO3- was used as a reference anion. In other experiments, Na' was replaced isosmotically by K+, Mg2+ or Ca2+. All flux media were freshly prepared every day and the osmolarity was kept constant at 295+5 mosM 1'. The tubes were capped and incubated at 37 °C for 0. 5, 10, 20 and 30 min. In control experiments we verified that external pH remained almost constant (±004 pH units) during the incubation period. To stop the reaction. tubes were chilled at 4 °( for 1 min and then centrifuged for 3 min at 1750 g at 4 'C. The supernatants were removed an(l the red cell pellet washed three times with 150 mm-NaNO3 or 150 mM-NaCl. At the end of the last wash, the red cell pellet was haemolyzed with 4 ml of 0-02 % Acationox (Monoject Scientific, St Louis, MO, USA). The tubes were centrifuged for 20 min at 5000 g. The supernatants were transferred into tubes for Cd2" analysis in an atomic absorption spectrophotometer (Carl Zeiss M4QIII, Oberkochen, Germany) and for measurement of haemoglobin absorbance at 541 nm. Cd2+ standards were prepared by dilution in Acationox, 002%. In control experiments we verified that the Cd2' readings did not undergo interference by the haemolysates (similar Cd2' readings were obtained by adding Cd2+ to distilled water or to Acationox-treated haemolysates). Figure 1 shows internal Cd2+ content as a function of time. The initial rate of Cd2+ uptake was calculated from the initial slope of this function. In all flux media. cell Cd2+ content increased linearly with time for at least 10-15 min. Therefore. fluxes were measured by using incubation times of 7-5-10 min. Figure 1 shows that DIDS was able to inhibit an important fraction of CId2+ influx. DIDS-sensitive Cd2+ uptake was taken as a marker of Cd2+ influx through the anion exchanger (for details on DIDS-sensitive cation fluxes see Becker & Duhm. 1978; Funder et al. 1978; Garay et al. 1986).
Measurement of HCO3- concentration in the flux media Capillary tubes were filled with aliquots of flux media, and were then sealed at both ends as a standard. Another set of capillaries was filled with the supernatants of the cell suspensions, taken at the end of the flux experiment. The capillaries were equilibrated for 10 min and the HCO3- concentrations were measured in an AVL-945 gas analyser (Instrumental Biomed, Graz, Austria). In control experiments we verified that values of HCO3- concentrations were stable (within +05%) for equilibration times between 0 and 30 min. In all cases, the pH of the flux media was measured by using a Ross electrode (Orion Research, Boston, MIA, USA). The measurements of HC03- concentrations and pH. before and after incubation with the cells, exhibited in almost all cases a variation less than 3%,. The mean values were used for kinetic analysis or represented in the figures. The effect of drugs To study the effect of drugs on red cell Cd2+ uptake, concentrated stock solutions of the compounds were prepared by dilution in water. ethanol or dimethyl sulphoxide. The cell suspensions (in C1--HCO3- media, Cd2+-free, containing C1- 130 mm, HC03- 15 mM) were preincubated for 3 min (at 4 °C) with different concentrations of compounds (final concentrations of ethanol and dimethyl sulphoxide did not modify ion transport per se). Finally, CdCl2 was added up to a final concentration of 100 /IM. Furosemide was obtained from Hoechst Laboratory (La Defense, Paris. France). Amiloride was a gift from Merck, Sharp & Dohme Laboratories (Paris. France). Bumetanide was obtained from Leo Laboratory (Vernouillet. France). DIDS, SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'disulphonic acid) and all other chemicals were either from Merck or Sigma (distributed through
Coger. Paris, France). RESULTS
Pharmacological properties of Cd2+ uptake in human red blood cells Several ion transport inhibitors were tested on the initial rate of red cell Cd2+ uptake (in bicarbonate and chloride medium containing CdCl2, 100 UtM). Figure 2 shows that DIDS inhibited about 95 % of Cd2+ uptake with an IC50 (concentration
CADMIUM TRANSPORT IN RED CELLS
127
100 0
80
-
0
0
o
60 0
X 40. 20
0
0
0-1
1 10 DIDS concentration (,M)
100
Fig. 2. Inhibition of red cell Cd2+ uptake by DIDS. It can be seen that DIDS was able to inhibit about 95 % of Cd2+ uptake with ICO of 0 3 /Mm. Fluxes were measured in Cl--HCO3medium containing 130 mm- Cl-, 15 mm- HCO3- and 100 ,tM-CdCl2. The same results were obtained in three other experiments. -C ., r-
0
2
E E
a, 0 +Cu
0)
ax
0 0-25 0-5 0.7 External Cd2+ concentration (mM)
-1.0 -0.75 -0.5 -0.25
Cl) C]
1-2 0.8 1-0 0.6 0-4 External Cd2+ concentration (mM) Fig. 3. DIDS-sensitive Cd2+ uptake as a function of external Cd2+ concentrations ([Cd2].). Cd2+ uptake reached saturation at high [Cd2+]0. Cd2+ uptake was measured in bicarbonate (2 mM) and chloride medium. Values represent means of triplicates (the range of variation was smaller than the size of the symbols). Inset, Hanes plot of the data. Apparent dissociation constant for [Cd2+]. (Kcd) was obtained from the intercept with the x-axis and Vmax Cd, the maximal rate of Cd2+ uptake, was obtained from the slope. KCd was about 1 mm and Vmax Cd about 4-3 mmol (1 cells)-' h-'. 0-0
0.2
giving 50 % of maximal inhibition) of 0-3 /tM. A similar extent of inhibition was obtained with SITS (IC50 = 0-3 /tM), xipamide (IC50 = 80,M), phloretin (IC50 = 100 /IM), and furosemide (IC50 = 500 LlM). Partial inhibition was obtained with high concentrations of bumetanide (700 ,UM) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) (120 /kM). Ouabain and amiloride were without inhibitory effect on Cd2+ uptake. This pharmacological pattern agrees well with that of anion carrier
-|
M. LOU, R. GARAY AND J. 0. ALDA
128 A r-
400
-c
_ 350* C.) _ 300 .5 E Zs. 250
0
/~~~~~~~~ 0
0
X 200
0
0
a
+A 150 a) 100 0)
50.
Cl c]
O
C
en 5
15 20 25 30 External HC03- (mM)
10
B
30
25
.i 0.
20
+
15
-
0
10
-6
-4
-2
4
6 External HCO3- (mM) 02
35
8
10
0
40
45
12
Fig. 4. A, DIDS-sensitive Cd2+ uptake as a function of external bicarbonate concentrations ([HCO3-]0) in nitrate medium. It can be seen that Cd24 uptake was: (i) stimulated by low [HCO3-]0 up to a maximal Cd2+ uptake of about 400 4umol (1 cells)-' h-' at [HCO3-]0 t 11 mm and (ii) inhibited by excess [HCO3-]j. Bicarbonate was replaced isosmotically by
nitrate. Similar results were obtained in three other experiments. B, Hanes plot of the first, stimulatory phase (bicarbonate concentrations from 0 to 10 mM). Apparent dissociation constant for [HCO3-0 (KbiC) was obtained from the intercept with the x-axis and Vmax,bic, the maximal rate of Cd2+ uptake, was obtained from the slope. Linearity was obtained with n = 1. In four different experiments we obtained KbiC = 5-7 + 0-4 mm and
Vmax,bic = 710±19,umol (1 cells)-' h-' (mean +S.D.). fluxes (Garay et al. 1986). Therefore, we equated Cd2+ fluxes catalysed by the anion carrier to those inhibited by 10 /IM of DIDS. Stimulation of Cd2+ uptake by external Cd2+
DIDS-sensitive Cd2+ uptake
was
measured in human red cells incubated in ([Cd2+]O).
bicarbonate and chloride medium containing different Cd2+ concentrations
CADMIUM TRANSPORT IN RED CELLS
129
A
1800 T 1600 1400 0
1200
-o
1000
0
/~~~0\ / \
X800 +
600
/ -
*> 400- O C)I 200 /
0
5
10
30 20 25 15 External HCO3- (mM)
35
40
45
15
20
10.
B 0. +
0
-20
-15
-10
-5
8-
66-
0
5
10
External HC03- (mM)
Fig. 5. A, DIDS-sensitive Cd'+ uptake as a function of external HCO3- concentrations ([HCO,-]0) in chloride medium (105 mM). Cd2+ uptake was qualitatively similar and higher than in chloride-free medium (Fig. 4A). Moreover, maximal Cd2+ uptake was found at [HCO.3-]o 15 mm. External HCO - was isosmotically replaced by NO3-. External Cd2 concentration was 100 /M. Similar results were obtained in four other experiments. B Hanes plot of the first, stimulatory phase (bicarbonate concentrations from 0 to 15 mM). 189+1-2mM and Vm..,ic= In five different experiments we obtained K -i,= 3860 +430 ,tmol (1 cells)-' h-l (mean + S.D.).
Figure 3 shows that in the 10-200 fM [Cd2+] range, Cd2+ uptake was almost a linear function of [Cd2+]O, and that high external Cd2+ concentrations were required to reach saturation. Indeed, a Hanes plot of the data (Fig. 3, inset) revealed an apparent dissociation constant for external Cd2+ (KCd) of about 1 mm and a maximal 5
PHY 443
M. LOU, R. GARAY AN,D J. 0. ALDA
130
rate (Vmax) of about 4 3 mmol (1 cells)-1 h-1 (for details of Hanes plots see Garay & Garrahan, 1973). In all following experiments we used flux media containing 100 ,am-Cd2".
Stimulation of Cd2+ uptake by external bicarbonate In studying the effect of bicarbonate on Cd21 uptake, we measured initial and final [HCO3j0 as described in Methods. Experiments where [HC03-j varied more than 5 % were rejected. s 2.0 n 1.8
a.)/
- 1.6
E 1 .4 E
1.2.
0.8/
+
~0*4 0~~~~~~~~~~~~~~ C_
$n o
0-
0
20
40 80 100 120 60 External Cl- concentration (mM)
140
160
Fig. 6. DIDS-sensitive Cd2+ uptake as a function of external chloride concentration ([Cl-]l). It can be seen that: (i) in the presence of 15 mM-bicarbonate (A), Cd2" uptake was strongly stimulated by increasing [Cl-]o following a non-saturable function and (ii) in media without added bicarbonate (0), [Cl-]0 had almost no effect on Cd2" uptake. External Cl- was isosmotically replaced by different N03- concentrations. External Cd2+ concentration was 100 /iM.
In chloride- and bicarbonate-free media (NO3- medium), DIDS-sensitive Cd2+ uptake was very small (less than 0 05 mmol (1 cells)-' h-'; contaminating bicarbonate from atmospheric CO2 was about 0 5 mm). Adding bicarbonate strongly stimulated Cd21 uptake, following a complex function. Figure 4A shows that the stimulation of Cd2+ uptake exhibited inhibition by excess [HCO3-j0, with a maximal Cd2+ uptake of about 400 /tmol (1 cells)-1 h-' at a bicarbonate concentration of about 11 mm. Figure 4B shows a Hanes plot of the first, stimulatory phase (between 0 and 10 mm [HCO3-j0). In four different experiments we obtained an apparent dissociation constant for [HCO3-j0 (KbiC) of 5*7 +0 4 mm (mean + S.D.) and Vmax = 710+ 19 ,tmol (1 cells)-' h-1. A more important stimulation by bicarbonate was obtained in chloride media. Figure 5A shows a maximal Cd2+ uptake of about 1700 jtmol (1 cells)-' h-1 at a bicarbonate concentration of about 16 mm. Figure 5B shows a Hanes plot of the first, stimulatory phase (between 0 and 15 mm [HCO3-j0). In five different
CADMIUM TRANSPORT IN RED CELLS
131
experiments we obtained an apparent dissociation constant for [HCO3-j0 (KbiC) of 18-9 + 1-2 mm (mean+ S.D.) and Vmax = 3860 + 430 /tmol (1 cells)-' h-1.
Stimulation of Cd2+ uptake by external chloride Figure 6 shows that: (i) in media without added bicarbonate, chloride ([Cl-]0) had almost no effect on DIDS-sensitive Cd2+ uptake (at [Cl-] = 145 mm, DIDS-sensitive , 2500 0
2250 @ 2000 o 1750
E
0
1500
1250 + 1000
, 750 ,, 500
c,
p/ s
250
o
6.0
6.2
6.4
6.6
6.8 7-0 7.2 External pH
7.4
7.6
7-8
8.0
Fig. 7. Stimulation of DIDS-sensitive Cd2+ uptake by alkalinization. Circles and squares indicate two different experiments. It can be seen that a change in pH from 6-2 to 7-8 stimulated Cd2+ uptake by more than one order of magnitude. The flux medium contained 130 mm of chloride and 15 mm of bicarbonate. External pH was changed by addition of MES, 2-(N-morpholino)ethanesulphonic acid (6-6 8), MOPS and Tris. External Cd2+ concentration was 100 fM.
Cd2+ uptake was lower than 80 jtmol (1 cells)-' h-1) and (ii) in the presence of bicarbonate, Cd2+ uptake was strongly stimulated by [Cl-]0. This stimulatory function did not reach saturation at physiological chloride concentrations (Fig. 6). Our data allowed us to estimate that under physiological chloride and bicarbonate concentrations and a cadmium concentration of 100 saM, DIDS-sensitive Cd2+ uptake should be ~ 2000 ,umol (1 cells)-' h-1. Stimulation of Cd2+ uptake by alkalinization DIDS-sensitive Cd2+ uptake exhibited profound stimulation by alkalinization. Figure 7 shows that a change in pH from 6-2 to 7-8 increases Cd2+ uptake by more than one order of magnitude. In addition, the data suggest that the inflexion of the stimulatory function was close to physiological values of pH (Fig. 7). Inhibition of Cd21 uptake by phosphate Figure 8A shows DIDS-sensitive Cd2+ uptake as a function of external phosphate, in media containing different bicarbonate concentrations. It can be seen that phosphate was a modest inhibitor of Cd21 uptake. In bicarbonate media, high 5-2
M. LOU, R. GARAY AND J. 0. ALDA
132
phosphate concentrations were required to inhibit Cd2+ uptake. Indeed, no more than 25% inhibition was observed at physiological external phosphate concentrations (Fig. 8A). Figure 8B shows a Dixon plot of the data. In five different experiments (in bicarbonate media) we obtained a Kpo4 (apparent dissociation constant for external phosphate) of 35 +04 mm (mean ±S.D.). A
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Fig. 8. A, inhibition of DIDS-sensitive Cd2+ uptake by external phosphate. Flux media contained different bicarbonate concentrations (Z = without added HCO3-, A = 7-5 mM-HCO3- and * = 15 mM-HCO3). It can be seen that high phosphate concentrations were required to inhibit Cd21 uptake. Flux media contained chloride as major monovalent anion. External Cd2+ concentration was 100 sm. Similar results were obtained in four other experiments. B, Dixon plot of the inhibition of Cd2+ uptake by phosphate (data from Fig. 8A). In five different experiments (in bicarbonate media) we obtained a KP04 (apparent dissociation constant for external phosphate) of 3.5 + 04 mM (mean +S.D.).
CADMIUM TRANSPORT IN RED CELLS
133
Other ions Sulphate was a very modest inhibitor of DIDS-sensitive Cd2+ uptake (it inhibited about 10 % of Cd2+ uptake at a concentration of 20 mM). Physiological concentrations of cations Na+, Mg2+ and Ca2+ were unable to modify Cd2+ uptake. In addition, Cd2+ 1200
a) 1000
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45
Fig. 9. Stimulation of DIDS-resistant red cell Cd2+ uptake by external bicarbonate. It can be seen that bicarbonate had more stimulatory action in nitrate (A) than in chloride (D) media.
uptake was insensitive to external K+ (isosmotic replacement of Na+ by K+ was unable to modify Cd2+ uptake).
Superoxide dismutase The anion carrier is able to transport superoxide anions. However, superoxide dismutase (SOD, 500 U ml-') was unable to modify DIDS-sensitive Cd2+ uptake.
DIDS-resistant red cell Cd2+ uptake DIDS-resistant Cd2+ uptake was resistant to ouabain, amiloride and phloretin and increased linearly with external Cd2+ concentration (data not shown). In addition, Fig. 9 shows that it was stimulated by [HCO3-j0 in nitrate media (a less important stimulation was observed in chloride media). Therefore, DIDS-resistant Cd2+ uptake in nitrate-bicarbonate media was higher than in chloride-bicarbonate media (about 50% vs. 5% in Fig. 2). Red cell Cd2+ efflux In contrast with Cd2+ uptake, the initial rate of Cd2+ efflux was undetectable and was independent of the presence or absence of external Ca2 . Indeed, in erythrocytes loaded up to 1 mmol (1 cells)-' of total cadmium content, the initial rate of Cd2+ efflux, if any, was less than 1 ,umol (1 cells)-' h-1.
134
M. LOU, R. GARAY AND J. 0. ALDA DISCUSSION
In bicarbonate-containing medium, red blood cells exhibit high Cd21 influx. For instance, Cd2+ fluxes in Figs 4 and 5 are similar in magnitude to the Na+ and K+ fluxes catalysed by the red cell Na+-K+ pump. This and the high sensitivity of the atomic absorption methods for cadmium (with standard error lower than 1-2 %) allowed us to precisely measure cadmium movements across human red cell membranes. Our values for Cd2+ uptake are higher than those previously obtained by Garty et al. (1986) in rat red blood cells. This may result from: (i) a difference between rat and human red blood cells and/or (ii) the fact that Garty et al. worked in Cl- medium without added HC03- Moreover, in contrast with 'Garty et al. (1986), we were unable to detect an initial rapid phase of Cd2+ uptake (Cd2+ influx was linear for more than 7 min). This can be explained by the fact that, unlike Garty et al. (1986), we have discarded the cell membranes. Therefore, the rapid phase of Cd2+ uptake may represent Cd2+ adsorption to the cell membrane. One technical problem in measuring red cell Cd2+ fluxes is to maintain constant medium HC03- concentrations. In media without HC03- added some is formed from atmospheric and metabolic (red cell-generated) CO2. In bicarbonate-containing media, some HC03- can be lost through CO2 evaporation. In order to avoid potential artifacts we capped the tubes during the flux experiment, we controlled the external pH and we measured systematically the initial and final external bicarbonate concentrations. Changes in external bicarbonate concentrations during the flux experiment were lower than 5-10 % of initial values. A screening of transport inhibitors showed that DIDS and furosemide inhibited a very important fraction of red cells Cd2+ uptake with IC50 similar to those classically obtained for anion carrier fluxes (for references see Gunn, 1979; Garay et al. 1986). Indeed, at physiological bicarbonate concentrations, DIDS-sensitive Cd2+ uptake was about 95 % of the total Cd2+ uptake. This is in line with Garty et al. (1986) who found that Cd2+ uptake in rat red blood cells was resistant to metabolic inhibitors, suggesting passive transport. Catalysis of Cd2+ uptake by the anion exchanger requires Cd2+ to be transported in the form of an anionic complex, particularly a monovalent anion. On the other hand, Cd2+ uptake was strongly dependent on the presence of bicarbonate. This suggests that the translocating anionic species has at least one bicarbonate ion. In addition, it can have one OH- ion because the pKD (-log of dissociation constant) for the Cd-OH+ ion pair is about 7-6 (Bernard & Busnot, 1984). Finally, chloride strongly stimulated bicarbonate-dependence Cd2+ uptake. Taken together these findings suggest that one translocating species can be the monovalent anion
complex:
[Cd(OH)HCO3Cl]-.
On the other hand, a significant, bicarbonate-dependent Cd2+ uptake was observed in N03- medium. Therefore, another translocating species can be the [Cd(OH)(HCO3)2] monovalent anion complex. However, we cannot exclude the possibility that other minor anionic cadmium complexes (Baes & Mesmer, 1976; Bernard & Busnot, 1984; Prince, 1987) such as
CADMIUM TRANSPORT IN RED CELLS 135 [Cd(HCO3)2Cl]-, [Cd(OH)CO3]-, [Cd(CO3)Cl]- and [Cd(OH)(Cl)2 can also be transported by the anion carrier. The red cell anion exchanger is able to catalyse much higher fluxes for monovalent than for divalent anions (Gunn, 1979). Therefore, the inhibition of Cd2+ uptake by excess bicarbonate may be interpreted as resulting from additional bicarbonate complexation, with the formation of divalent anionic species such as [Cd(OH)-
(HCO3)3]2-, [Cd(CO3)2]2- or [Cd(OH)(HCO3)2C1]2-. Regarding the pH dependence, DIDS-sensitive Cd2+ uptake exhibited the opposite tendency to that previously reported for anion carrier fluxes (Gunn, 1979), i.e. it was stimulated by alkalinization (from pH 7-0 to 7 8). This can be explained by an increase in the concentration of the translocating species [Cd(OH)HCO3Cl]- and [Cd(OH)2HCO3)]-, due to the increase in OH- concentration. Besides bicarbonate and chloride, phosphate was a physiological anion able to modify cadmium uptake. Modest inhibition of cadmium uptake by physiological concentrations of phosphate may be explained by formation of a non-translocating species such as Cd3(PO4)2. Red cell Cd2+ uptake may have important toxicological consequences. Thus, cadmium exposure from dietary sources (30-50 jtg day-'; Yost, 1984) results in continuous absorption from ingested food into blood where it is transported and delivered to target tissues. Blood cadmium reaction can be categorized into two compartments: (i) a rapid one, clearing about 99 % of acutely administered cadmium within 3 h and (ii) a slow one in red blood cells, accounting for chronic cadmium exposure (Garty et al. 1981). Our results suggest that the slow exchanges between red cells and plasma are mediated by the anion carrier. However, it is important to note that: (i) red cell Cd2+ uptake in vivo is strongly limited by the extent of cadmium bound to proteins and (ii) rate constants of outward Cd2+ movements were undetectable, suggesting that haemoglobin and/or other intracellular proteins bind Cd2+ with high affinity and high capacity. It appears therefore that determination of in vivo Cd2+ uptake requires further investigation. Besides erythrocytes, the anion carrier appears to exist in the kidney, where it may participate in bicarbonate reabsorption (Schild, Giebisch, Karniski & Aronson, 1986). Whether the anion carrier is involved in the renal handling of cadmium deserves further investigation. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HCO3- such as [Cd(OH)(HCO3)2]- and [Cd(OH)(HCO3)Cl]-. This study was supported by the Ion Transport and Hypertension Association, France. REFERENCES
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