- Email: [email protected]
Intracellular calcium mobilization in rat platelets is adversely affected by copper deficiency
Biochimica et Biophysica Acta, 1175 (1993) 263-268 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00
263
BBAMCR 13310
Intracellular calcium mobilization in rat platelets is adversely affected by copper deficiency W. Thomas Johnson and Steven N. Dufault United States Department of Agricuhure, Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND (USA)
(Received 14 February 1992) (Revised manuscript received 31 August 1992)
Key words: Platelet; Cytosolic calcium; Copper deficiency; Thrombin; Fura-2; (Rat)
The influence of copper deficiency on the mobilization of Ca 2÷ from intracellular stores following ionomycin treatment or thrombin activation of rat platelets was examined using the fluorescent indicator, fura-2, to measure changes in cytosolic Ca 2÷ concentration ([Ca2+]i). Platelets, obtained from copper-deficient and control rats and loaded with fura-2, were suspended in medium containing 1 mM EGTA and no added Ca 2+. The size of the internal Ca 2+ pools in the suspended platelets was estimated from the rise in [Ca 2+ ]i following maximal discharge of stored Ca 2÷ by treatment with 1/x M ionomycin. Peak [Ca 2÷ ]~ following ionomycin treatment was lower in platelets from copper-deficient rats compared to control rats (148 + 27 nM vs. 188 + 17 nM), suggesting that the size of the Ca 2+ storage pools was decreased by copper deficiency. Furthermore, once internal Ca 2÷ stores were discharged by ionomycin, [Ca2+]i remained elevated in platelets from copper-deficient rats, but decreased in control rats. These data indicate that copper deficiency may inhibit the efflux of Ca 2+ from platelets after its release from internal stores by ionomycin treatment. In platelets from copper-deficient and control rats, stimulation with 0.1 U / m l thrombin led to rapid rise followed by a slow decay in [Ca2+]i . However, peak [Ca2+]i was lower in platelets from copper-deficient rats than in control rats (94 + 19 nM vs. 131 + 16 nM). These findings imply that by reducing the amount of Ca 2+ available for release from intracellular stores, copper deficiency also reduces [Ca2+]i following thrombin activation in the absence of external Ca 2+.
Introduction Thrombin, a potent agonist of platelet activation, elicits a variety of physiologic responses such as aggregation, shape change and the secretion of granule contents [1]. In rats, deprivation of dietary copper produces several aberrations in thrombin-induced platelet responses. C o m p a r e d to platelets obtained from rats fed adequate copper, platelets from copperdeficient rats upon thrombin stimulation undergo enhanced actin polymerization and accumulate greater amounts of cytoskeletal myosin [2]. The rate of dense granule secretion also is increased 2-fold in thrombinactivated platelets from copper-deficient rats [2,3]. Although these findings indicate that copper has a function in maintaining normal platelet responses to thrombin, the biochemical mechanisms for the changes in platelet responsiveness during copper deficiency are
Correspondence to: W.T. Johnson, USDA, ARS, GFHNRC, P.O. Box 7166, University Station, Grand Forks, ND 58202. USA.
not well understood. Recently, it was found that in thrombin-activated platelets, copper deficiency impairs phosphorylation of the 40 k D a protein substrate for protein kinase C, and increases the susceptibility of dense granule secretion to inhibition by H-7, an inhibitor of protein kinase C [3]. Thus, one way that copper deficiency may affect thrombin-induced platelet responses is by modifying the regulation of signal transduction by protein kinase C. It is generally accepted that a change in cytosolic free Ca 2÷ concentration ([Ca2+] i) is a requirement for the activation of many platelet functions [4,5]. Following thrombin stimulation of platelets, a rise in [Ca2+]~ occurs [6-9] that may modulate several Ca2+-depen dent cellular processes. The increased [Ca2+]i in activated platelets may regulate the polymerization of actin [10] possibly by activating the nucleating, severing or capping activities of actin-binding proteins such as gelsolin [11,12]. The rise in [Ca2+] i may also promote the phosphorylation of myosin and its association with the platelet cytoskeleton by activating calmodulin-dependent myosin light chain kinase [10,13]. Protein kinase C
264 also requires Ca 2+ for full activation [14,15]. The activation of myosin light chain kinase and protein kinase C is a critical element of stimulus-response coupling in platelets and both kinases have been implicated in the mechanism for dense granule secretion from stimulated platelets [14,16-18]. Thus, dense granule secretion, through its dependence on myosin light chain kinase and protein kinase C, also has a requirement for C a 2 +.
An increase in [Ca2+] i is a central feature that is common to all the thrombin-induced platelet responses affected by copper deficiency, i.e., actin polymerization, myosin-actin interaction, dense granule secretion and protein kinase C activation [2,3]. It is possible, therefore, that the altered responses of thrombinactivated platelets during copper deficiency are caused by perturbations of the thrombin-induced rise in [Ca2+] i. Furthermore, the past studies showing the effects of copper deficiency on platelet responses used washed cells in the absence of extracellular Ca 2÷ [2,3]. This suggests that any influence of copper deficiency on Ca 2÷ flux following thrombin activation may be on the release of Ca 2+ from intracellular stores or on its efflux out of the cell. Accordingly, we present here experiments using fura-2-1oaded platelets suspended in the absence of extracellular Ca 2+ to examine the effect of copper deficiency on [Ca2+] i following thrombin stimulation. Materials and Methods
Animals and diets Male weanling Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) 1 were housed in individual stainless steel cages in a temperature- and humidity-controlled room with a 12-h light/dark cycle. These animals were divided into two groups with each containing six animals. One group was fed a copper-deficient diet ( < 0.5/zg C u / g diet) and the other a diet containing adequate copper (5.9 /zg C u / g diet). The initial mean weights + S.D. of rats in these groups were 81.2 + 10.2 g and 81.1 + 9.1 g, respectively. The diets were composed of 940.0 g of casein-based copper- and iron-free basal diet (No. TD84469, Teklad Test Diets, Madison, WI), 50.0 g safflower oil (Teklad Test Diets) and 10.0 g of Cu-Fe mineral mix per kg diet. Copperdeficient and copper-adequate diets were obtained by using Cu-Fe mineral mixes containing either no added Cu or 0.5 g of Cu per kg mix as previously described [2,19]. The rats were fed these diets and distilled,
1 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States D e p a r t m e n t of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
deionized water (Super-Q Water System, Millipore Corporation, Bedford, MA) ad libitum for 5 weeks.
Preparation of platelets Following ether anesthesia, 6-8 ml of blood were withdrawn from the vena cava of rats into anticoagulant solution (0.16 ml/ml blood) containing 0.11 M glucose, 0.085 M sodium citrate and 0.071 M citric acid. The blood was centrifuged (160 × g , 20 min, 25°C) to obtain the platelet-rich plasma. Platelets were then obtained by centrifuging (730 × g, 10 min, 25°C) the platelet-rich plasma [20]. The platelet pellet was resuspended to 5 • l0 s platelets/ml in physiologic saline containing 145 mM NaCI, 5 mM KC1, 1 mM MgSO 4 • 7H20, 10 mM Hepes, 10 mM D-glucose (pH 7.4). As determined by electronic cell counting, the platelet suspension contained less than 0.01% red blood ceils and less than 0.001% of leukocytes. Platelets were then loaded with fura-2 by incubating the cell suspension in the presence of 5 /zM fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 45 min at 37°C. The platelet suspension was then centrifuged (730 x g, 10 min, 25°C) and washed once before final suspension at 5" 108 cells/ml in the physiological saline solution described above.
Fluorescence measurements An aliquot (0.2 ml) of suspension containing fura-2 loaded platelets was added to 37°C buffer (1.8 ml) containing 145 mM NaC1, 5 mM KC1, 1 mM MgSO 4 • 7H20, 10 mM Hepes, 10 mM D-glucose, 1.1 mM E G T A (pH 7.4) to give a final platelet concentration of 1 • 10S/ml. After a 1 min incubation, either ionomycin (1 /xM) (Calbiochem, San Diego, CA) or rat thrombin (0.1 U / m l ) (Sigma, St. Louis, MO) was added and fluorescence was measured at the emission wavelength of 510 nm with the excitation wavelength switched continuously between 340 and 380 nm (RF-5000 Spectrofluorometer, Shimadzu Corp.). Fluoresence measurements were carried out at 37°C. The ratio of the fluorescence intensities at the two excitation wavelengths was used to determine [Ca2+] i [21,22]. Calibration of [Ca2]i was by lysing the cells with 50 /zM digitonin in the presence of 1 mM CaC12 or 10 mM EGTA (pH 9.0) [23].
Other determinations Livers were removed and perfused with cold 0.15 M NaC1 to remove trapped blood. Identical lobes from each liver were ashed in a muffle furnace for 48 h at 450°C. The residue was then dissolved in 1.0 ml of concentrated HNO 3 (double distilled, GFS Chemicals, Columbus, OH) and returned to the muffle furnace for an additional 24 h. The remaining residue was dissolved in 2.0 ml of concentrated HC1 (reagent grade, Fisher Scientific, Fair Lawn, N J), diluted and analyzed
265 TABLE I
Indices o f copper status in rats fed copper deficient (Cu - ) or copper adequate (Cu + ) diets Liver and concentrations are based on the weight of the lyophilized liver sample. A unit of ceruloplasmin activity is that amount which catalyzes the oxidation of 1 p.mol o-dianisidine/min. Hematocrit and hemoglobin concentration were determined on whole blood using an electronic cell counter. Values are m e a n s + S.D.
Diet CuCu+
n Liver Cu (p.mol/kg)
Ceruloplasmin Hematocrit (units/l plasma) (fractional)
6 15.2+_ 4.3 a 0.4+- 0.6 a 6 150.7+-13.3 61.7_+32.7
Hemoglobin (g/l)
0.18+0.03 a 52_+11 a 0.42+-0.02 138+- 9
CuD
CuA
0 t~ LId 0 m LI.I
140nM-96nM
--
..~ n.-
70nM--
rV~l '
'
Thb
Thb
I
I
60s
Values for rats fed C u - are significantly lower than those for rats fed C u + ( P < 0.05, Student's t-test).
a
for and by flame atomic absorption spectroscopy [2]. Hematocrit, hemoglobin concentration and amine oxidase activity of plasma ceruloplasmin were determined as described in a previous report [2]. Data were analyzed statistically by analysis of variance (ANOVA) followed by Tukey's test for comparison between individual means or, when appropriate, by Student's t-test ( S A S / S T A T Version 6, SAS Institute, Cary, NC). Results
As shown in Table I, rats consuming diets containing low copper developed anemia and had significantly lower liver copper and plasma ceruloplasmin activity than their counterparts consuming diets containing adequate copper. These signs are characteristic of poor copper status [24-26] and confirm that the low copper diet induced copper deficiency in the rats consuming it. Fig. 1 shows examples of the change in [Ca2+] i that was induced by ionomycin treatment. Ionomycin (1
CuA
CuD
o
o lad .9 0 re"
Thb
10n0 I
I
60 s
?
t
Thb
Iono I
I
60s
Fig. 1. The release of internally stored Ca 2+ by ionomycin and subsequent stimulation of Ca 2÷ efflux by thrombin in platelets from copper-deficient (CUD) and control (CuA) rats. Platelets were suspended in the presence of 1.0 m M E G T A and stimulated with 1 p,M ionomycin (Iono) followed by 0.1 U / m l of thrombin (Thb). The ratio of fura-2 fluorescence signals at 340 n m and 380 n m excitation was used to calculate cytosolic Ca 2+ concentrations.
~
I
I
60s
Fig. 2. Changes in [Ca ~+ ]i following thrombin activation of platelets
from copper-deficient (CUD) and control (CuA) rats. Platelets were suspended in the presence of 1 mM EGTA and activated with 0.1 U/ml thrombin (Thb). The ratio of the fluorescence signals at 340 nm and 380 nm excitation was used to calculate cytosolic Ca 2+ concentrations. Fluorescenceemission was measured at 510 nm.
/xM), which in the presence of E G T A translocates internally stored Ca 2÷ to the cytosol [27,28], caused [Ca2+] i to rapidly increase in platelets from copper-deficient and control rats. In these particular examples, the maximum [Ca2+]i induced by ionomycin was less in platelets from the copper-deficient rat (152 nM) than in platelets from the control rat (210 nM). However, [Ca2÷]i in ionomycin-treated platelets from the copper-deficient rat remained elevated until the platelets were subsequently stimulated with thrombin (1 U / m l ) . In contrast, [Ca2+] i in ionomycin-treated platelets from the control rat declined after the initial rise. Generally, for all animals in the experiment, [Ca2+]i was essentially constant in ionomycin-treated platelets from copper-deficient rats, decreasing at a rate of only 3 + 3 n M / m i n , but steadily declined in ionomycin-treated platelets from control rats during the interval between peak [Ca2+]i and the addition of thrombin. After thrombin stimulation, the rate of [Ca2÷]i decline in platelets from copper-deficient rats (47_+ 17 n M / m i n ) became identical to the rate in platelets from control rats (42 + 15 n M / m i n ) . As shown in Fig. 2, thrombin (0.1 U / m l ) activation of platelets from copper-deficient and control rats caused a rapid rise i n [ C a 2 + ] i , which returned to resting concentrations within 60 s after stimulation. In these examples, the maximum [Ca2+] i induced by thrombin stimulation was lower in platelets from copper-deficient rats (96 nM) than in platelets from control rats (140 nM). The examples shown in Figs. 1 and 2 suggest that peak [Ca2+] i following either thrombin activation or ionomycin treatment may be lower in platelets from copper-deficient compared to control rats. Table II summarizes the effects of copper status, thrombin activation and ionomycin treatment on peak [Ca2+] i in
266 T A B L E II The effects o f dietary copper, thrombin activation and ionomycin treatment on platelet cytosolic Ca 2 + concentration The ratio of fura-2 fluorescence at 340 n m and 380 n m excitation was used to calculate [Ca 2+ li prior to and following treatment of platelets with 1 U / m l thrombin and 1 /xM ionomycin. [Ca 2+ ]i in stimulated platelets are peak concentrations. Values are m e a n s ± S.D. Diet
n
Cu Cu+
6 6
[Ca 2 + ]i (nM) resting
thrombin
Ionomycin
65 ± 16 77± 9
94_+ 19 a 131_+16
148 ± 27 ab 188±17b
a The stimulus, whether thrombin or ionomycin, produced lower [Ca2÷ ]i in platelets from rats fed C u - ( P < 0.05, Tukey's test). b Ionomycin treatment produced higher [Ca 2÷ ]i than thrombin activation regardless of dietary copper content ( P < 0.05, Tukey's test). Dietary copper did not significantly affect resting [Ca 2+]i ( P > 0.05, Tukey's test).
platelets from all copper-deficient and control rats. Analysis of these data by ANOVA indicated that changes in [Ca2+]i following treatment with either thrombin or ionomycin depended on copper status (treatment x status interaction, P < 0.05) and which activator was used (treatment × activator interaction, P < 0.05). Prior to thrombin stimulation, [ C a 2 + ] i w a s similar in platelets from copper-deficient and control rats. Following thrombin stimulation, peak [Ca2+] i was less in platelets from copper-deficient rats compared to platelets from control rats. P e a k [Ca2+]i also was less following ionomycin treatment of platelets from copper-deficient rats than platelets from control rats. Regardless of whether thrombin or ionomycin was used as an activator, peak [Ca2+] i was less in platelets from copper-deficient compared to control. Furthermore, peak [Ca 2+]i was higher in ionomycin-treated platelets
O
o,q. u.i o
80 n M- -
Thb
[ono
I
I
60s
Fig. 3. C h a n g e s in [Ca z+ ]i in platelets from a copper-deficient rat stimulated with 0.1 U / m l thrombin (Thb) followed by treatment with 1 g M ionomycin (Iono). T h e fluorescence ratio was used to calculate cytosolic Ca 2+ concentrations as in Figs. 1 and 2.
than in thrombin-activated platelets regardless of copper status. Because [Ca2+]i tended to remain elevated in ionomycin-treated platelets from copper-deficient rats until further treatment with thrombin (see Fig. 1), it was of interest to determine if [Ca2+] i would remain elevated following ionomycin treatment if the platelets were first activated by thrombin. As shown in Fig. 3, thrombin activation of platelets from a copper-deficient rat caused the usual rise and decay of [Ca2+] i. Additional treatment with ionomycin caused a second rise in [Ca2+] i which subsequently declined to the initial concentration. This result was typical of platelet samples from copper-deficient rats. Discussion
Past investigations have shown that deprivation of dietary copper alters cytoskeletal remodeling, accelerates dense granule secretion and may impair protein kinase C activation in thrombin-activated rat platelets [2,3]. Results from the present study suggest that copper deficiency also alters the thrombin-induced release of Ca 2+ from intracellular stores by reducing the size of these stores. In the presence of EGTA, treatment of platelets with 1 /~M ionomycin translocates internally stored Ca 2+ to the cytosol and prevents its resequestration into the storage pools [27,28]. Because ionomycin treatment maximally discharges the thrombin-sensitive Ca 2÷ pool [27], the size of this pool can be estimated from [Ca2+] i measured in platelets following ionomycin treatment [9]. In our study, peak [Ca2+] i in ionomycintreated platelets from control rats was 188 + 17 nM, which is similar to the previously reported value of 219 + 15 nM [9]. However, the peak lEa2+] i achieved following ionomycin treatment was significantly lowered by copper deficiency to 148 + 27 nM. Also, no further increases in [Ca2+] i occurred when ionomycintreated platelets from either copper-deficient or control rats were activated by thrombin, indicating that the thrombin-sensitive internal storage pools for Ca 2÷ were discharged by ionomycin and that no resequestration took place. Thus, it can be concluded that the amount of internally stored Ca 2+ in platelets is reduced by copper deficiency. Thrombin activation caused lEa2+] i to increase in platelets from copper-deficient and control rats. The peak [Ca 2÷ ]i following thrombin stimulation of platelets from control rats was 131 + 16 nM, which is comparable to the 161 + 6 nM previously reported for thrombin-activated rat platelets using similar experimental conditions [9]. However, the peak [Ca2+] i achieved following thrombin activation of platelets from copper-deficient rats was reduced to 94 + 20 nM. Because these experiments were performed with washed
267 platelets in the absence of extracellular Ca 2÷ and in the presence of E G T A , the increase in [Ca2+]i observed following thrombin activation of platelets from control and copper-deficient rats most likely represents release of Ca 2÷ from intracellular stores. However, as discussed above, copper deficiency reduces the size of the Ca 2÷ storage pool. Therefore, the rise in [Ca2+]i following thrombin activation of platelets from copper-deficient rats may be inhibited because less Ca 2÷ is available for release from internal stores. The decay of [Ca2+] i observed in ionomycin-treated platelets from control rats in the present study is consistent with previous results from ionomycin-treated human platelets [27-29]. Since the continued presence of ionomycin prevents resequestration of released Ca 2÷ back into the storage pools and does not contribute significantly to the efflux of Ca 2÷ from the cell, the decay of [Ca2+] i following ionomycin treatment in the presence of E G T A can be attributed to an active extrusion mechanism for Ca 2+ from the platelets [27,28]. After initially rising following ionomycin treatment, [Ca 2+]i remained essentially constant in platelets from copper-deficient rats. This implies that copper deficiency affects a mechanism or mechanisms responsible for transporting Ca 2+ out of platelets. However, thrombin activation subsequent to ionomycin treatment caused [Ca2+] i to decline at the same rate in platelets from copper-deficient and control rats. These observations indicate that copper deficiency may impair a constitutive, receptor-independent mechanism for the extrusion of Ca 2+ from platelets but has little effect on receptor-operated mechanisms for Ca 2+ efflux. Furthermore, once platelets from copper-deficient rats were activated by thrombin, their ability to remove Ca 2÷ released into the cytosol from internal stores by ionomycin treatment was not affected. This suggests that copper deficiency has little effect on the processing of Ca 2+ mobilized from internal stores once thrombin activation has taken place. It is not possible to address the nature of the Ca 2÷ transport mechanisms in rat platelets using results from the present study. However, human platelets contain at least two biochemically distinct Ca2+-ATPases in the plasma and intracellular membranes [30,31]. It is possible, therefore, that rat platelets also contain multiple Ca2+-ATPases, some of which are not receptor operated but provide a means for Ca 2+ transport into intracellular stores or out of the cell to help maintain Ca 2+ homeostasis. Inactivation of such a Ca2+-ATPase could explain the lower Ca 2+ stores in platelets from copper-deficient rats and also the inhibition of Ca 2+ efflux from these cells once the intracellular stores are discharged with ionomycin. The findings of the present study indicate that copper deficiency reduces the peak [Ca2+]i in platelets following thrombin activation but does not impair re-
ceptor-operated mechanisms for lowering [Ca2+]i once Ca 2+ is mobilized from internal stores. However, increased or sustained [Ca2+]i would be required to directly enhance thrombin-induced actin polymerization and myosin-actin interaction. Thus, the changes caused by copper deficiency on Ca 2+ mobilization from internal stores, which tends to lower [Ca2+]i in the absence of extracellular Ca 2÷, cannot explain the increase in actin polymerization and myosin association with actin that occurs in thrombin-stimulated platelets during copper deficiency. However, lower [Ca2+]i may impair Ca2+-activated mechanisms that negatively control these platelet responses, leading to their increase during copper deficiency.
Acknowledgements The authors would like to thank Pam Sakkinen for technical assistance and Lu Ann Johnson for performing statistical analysis of our data.
References 1 Holmsen, H. (1989) Ann. Med. 21, 23-30. 2 Johnson, W.T. and Dufault, S.N. (1989) J. Nutr. 119, 1404-1410. 3 Johnson, W.T. and Dufault, S.N. (1991) J. Nutr. Biochem. 2, 663-670. 4 Siess, W. (1989) Physiol. Rev. 69, 58-178. 5 Zucker, M.B. and Nachmias, V.T. (1985) Arteriosclerosis 5, 2-18. 6 Rink, T.J., Smith, S.W. and Tsein, R.Y. (1982) FEBS Lett. 148, 21-26. 7 Wenche, J. and Haynes, D.H. (1987) Biochim. Biophys. Acta 929, 88-102. 8 Thompson, N.T. and Scrutton, M.C. (1985) Eur. J. Biochem. 147, 421-427. 9 Heemslerk, J.W.M., Feijge, M.A.H., Reitman, E. and Hornstra, G. (1991) FEBS Len. 284, 223-226. 10 Fox, J.E.B. and Phillips, D.R. (1983) Semin. Hematol. 20, 243260. 11 Stossel, T.P. (1989) J. Biol. Chem. 264, 18261-18264. 12 Janmey, P.A., Chaponnier, C., Lind, S.E., Zaner, K.S., Stossel, T.P. and Yin, Y.L. (1985) Biochemistry 24, 3714-3723. 13 Fox, J.E.B. and Phillips, D.R. (1982) J. Biol. Chem. 257, 41204126. 14 Nishizuka, Y. (1984) Nature 308, 693-698. 15 Bell, R.M. (1986) Cell 45, 631-632. 16 Nishikawa, M., Tanaka, T. and Hidaka, H. (1980) Nature 287, 863-865. 17 Daniel, J.L., Holmsen, H. and Adelstein, R.S. (1977) Thrombos. Haemostas. 38, 984-989. 18 Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fijikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704. 19 Johnson, W.T. and Kramer, T.R. (1987) J. Nutr. 117, 1085-1090. 20 Jennings, L.K., Fox, J.E.B., Edwards, H.H. and Phillips, D.R. (1981) J. Biol. Chem. 256, 6927-6932. 21 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. 22 Cobbold, P.H. and Rink, T.J. (1987) Biochem. J. 248, 313-328. 23 Schaeffer,J. and Blaustein, M.P. (1989) Cell Calcium 10, 101-113. 24 Owen, C.A., Jr. and Hazelrig, J.B. (1968) Am. J. Physiol. 215, 334-338. 25 Alfaro, B. and Heaton, F.W. (1973) Br. J. Nutr. 29, 73-85.
268 26 Evans, J.L. and Abraham, P.A. (1973) J. Nutr. 103, 196-201. 27 Pollock, W.K., Sage, S.O, and Rink, T.J. (1987) FEBS Lett. 2, 132-136. 28 Johansson, J.S. and Haynes, D.H. (1988) J. Membrane Biol. 104, 147-163.
29 Rink, T.R. and Sage, S.O. (1987) J. Physiol. 393, 513-524. 30 Enouf, J., Bredoux, R., Bourdeau, N. and Levy-Toledano, S. (1987) J. Biol. Chem, 262, 9293-9297. 31 Enouf, J., Bredoux, R., Bourdeau, N., Sarkadi, B. and LevyToledano, S. (1989) Biochem. J. 263, 547-552.
263
BBAMCR 13310
Intracellular calcium mobilization in rat platelets is adversely affected by copper deficiency W. Thomas Johnson and Steven N. Dufault United States Department of Agricuhure, Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND (USA)
(Received 14 February 1992) (Revised manuscript received 31 August 1992)
Key words: Platelet; Cytosolic calcium; Copper deficiency; Thrombin; Fura-2; (Rat)
The influence of copper deficiency on the mobilization of Ca 2÷ from intracellular stores following ionomycin treatment or thrombin activation of rat platelets was examined using the fluorescent indicator, fura-2, to measure changes in cytosolic Ca 2÷ concentration ([Ca2+]i). Platelets, obtained from copper-deficient and control rats and loaded with fura-2, were suspended in medium containing 1 mM EGTA and no added Ca 2+. The size of the internal Ca 2+ pools in the suspended platelets was estimated from the rise in [Ca 2+ ]i following maximal discharge of stored Ca 2÷ by treatment with 1/x M ionomycin. Peak [Ca 2÷ ]~ following ionomycin treatment was lower in platelets from copper-deficient rats compared to control rats (148 + 27 nM vs. 188 + 17 nM), suggesting that the size of the Ca 2+ storage pools was decreased by copper deficiency. Furthermore, once internal Ca 2÷ stores were discharged by ionomycin, [Ca2+]i remained elevated in platelets from copper-deficient rats, but decreased in control rats. These data indicate that copper deficiency may inhibit the efflux of Ca 2+ from platelets after its release from internal stores by ionomycin treatment. In platelets from copper-deficient and control rats, stimulation with 0.1 U / m l thrombin led to rapid rise followed by a slow decay in [Ca2+]i . However, peak [Ca2+]i was lower in platelets from copper-deficient rats than in control rats (94 + 19 nM vs. 131 + 16 nM). These findings imply that by reducing the amount of Ca 2+ available for release from intracellular stores, copper deficiency also reduces [Ca2+]i following thrombin activation in the absence of external Ca 2+.
Introduction Thrombin, a potent agonist of platelet activation, elicits a variety of physiologic responses such as aggregation, shape change and the secretion of granule contents [1]. In rats, deprivation of dietary copper produces several aberrations in thrombin-induced platelet responses. C o m p a r e d to platelets obtained from rats fed adequate copper, platelets from copperdeficient rats upon thrombin stimulation undergo enhanced actin polymerization and accumulate greater amounts of cytoskeletal myosin [2]. The rate of dense granule secretion also is increased 2-fold in thrombinactivated platelets from copper-deficient rats [2,3]. Although these findings indicate that copper has a function in maintaining normal platelet responses to thrombin, the biochemical mechanisms for the changes in platelet responsiveness during copper deficiency are
Correspondence to: W.T. Johnson, USDA, ARS, GFHNRC, P.O. Box 7166, University Station, Grand Forks, ND 58202. USA.
not well understood. Recently, it was found that in thrombin-activated platelets, copper deficiency impairs phosphorylation of the 40 k D a protein substrate for protein kinase C, and increases the susceptibility of dense granule secretion to inhibition by H-7, an inhibitor of protein kinase C [3]. Thus, one way that copper deficiency may affect thrombin-induced platelet responses is by modifying the regulation of signal transduction by protein kinase C. It is generally accepted that a change in cytosolic free Ca 2÷ concentration ([Ca2+] i) is a requirement for the activation of many platelet functions [4,5]. Following thrombin stimulation of platelets, a rise in [Ca2+]~ occurs [6-9] that may modulate several Ca2+-depen dent cellular processes. The increased [Ca2+]i in activated platelets may regulate the polymerization of actin [10] possibly by activating the nucleating, severing or capping activities of actin-binding proteins such as gelsolin [11,12]. The rise in [Ca2+] i may also promote the phosphorylation of myosin and its association with the platelet cytoskeleton by activating calmodulin-dependent myosin light chain kinase [10,13]. Protein kinase C
264 also requires Ca 2+ for full activation [14,15]. The activation of myosin light chain kinase and protein kinase C is a critical element of stimulus-response coupling in platelets and both kinases have been implicated in the mechanism for dense granule secretion from stimulated platelets [14,16-18]. Thus, dense granule secretion, through its dependence on myosin light chain kinase and protein kinase C, also has a requirement for C a 2 +.
An increase in [Ca2+] i is a central feature that is common to all the thrombin-induced platelet responses affected by copper deficiency, i.e., actin polymerization, myosin-actin interaction, dense granule secretion and protein kinase C activation [2,3]. It is possible, therefore, that the altered responses of thrombinactivated platelets during copper deficiency are caused by perturbations of the thrombin-induced rise in [Ca2+] i. Furthermore, the past studies showing the effects of copper deficiency on platelet responses used washed cells in the absence of extracellular Ca 2÷ [2,3]. This suggests that any influence of copper deficiency on Ca 2÷ flux following thrombin activation may be on the release of Ca 2+ from intracellular stores or on its efflux out of the cell. Accordingly, we present here experiments using fura-2-1oaded platelets suspended in the absence of extracellular Ca 2+ to examine the effect of copper deficiency on [Ca2+] i following thrombin stimulation. Materials and Methods
Animals and diets Male weanling Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) 1 were housed in individual stainless steel cages in a temperature- and humidity-controlled room with a 12-h light/dark cycle. These animals were divided into two groups with each containing six animals. One group was fed a copper-deficient diet ( < 0.5/zg C u / g diet) and the other a diet containing adequate copper (5.9 /zg C u / g diet). The initial mean weights + S.D. of rats in these groups were 81.2 + 10.2 g and 81.1 + 9.1 g, respectively. The diets were composed of 940.0 g of casein-based copper- and iron-free basal diet (No. TD84469, Teklad Test Diets, Madison, WI), 50.0 g safflower oil (Teklad Test Diets) and 10.0 g of Cu-Fe mineral mix per kg diet. Copperdeficient and copper-adequate diets were obtained by using Cu-Fe mineral mixes containing either no added Cu or 0.5 g of Cu per kg mix as previously described [2,19]. The rats were fed these diets and distilled,
1 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States D e p a r t m e n t of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
deionized water (Super-Q Water System, Millipore Corporation, Bedford, MA) ad libitum for 5 weeks.
Preparation of platelets Following ether anesthesia, 6-8 ml of blood were withdrawn from the vena cava of rats into anticoagulant solution (0.16 ml/ml blood) containing 0.11 M glucose, 0.085 M sodium citrate and 0.071 M citric acid. The blood was centrifuged (160 × g , 20 min, 25°C) to obtain the platelet-rich plasma. Platelets were then obtained by centrifuging (730 × g, 10 min, 25°C) the platelet-rich plasma [20]. The platelet pellet was resuspended to 5 • l0 s platelets/ml in physiologic saline containing 145 mM NaCI, 5 mM KC1, 1 mM MgSO 4 • 7H20, 10 mM Hepes, 10 mM D-glucose (pH 7.4). As determined by electronic cell counting, the platelet suspension contained less than 0.01% red blood ceils and less than 0.001% of leukocytes. Platelets were then loaded with fura-2 by incubating the cell suspension in the presence of 5 /zM fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 45 min at 37°C. The platelet suspension was then centrifuged (730 x g, 10 min, 25°C) and washed once before final suspension at 5" 108 cells/ml in the physiological saline solution described above.
Fluorescence measurements An aliquot (0.2 ml) of suspension containing fura-2 loaded platelets was added to 37°C buffer (1.8 ml) containing 145 mM NaC1, 5 mM KC1, 1 mM MgSO 4 • 7H20, 10 mM Hepes, 10 mM D-glucose, 1.1 mM E G T A (pH 7.4) to give a final platelet concentration of 1 • 10S/ml. After a 1 min incubation, either ionomycin (1 /xM) (Calbiochem, San Diego, CA) or rat thrombin (0.1 U / m l ) (Sigma, St. Louis, MO) was added and fluorescence was measured at the emission wavelength of 510 nm with the excitation wavelength switched continuously between 340 and 380 nm (RF-5000 Spectrofluorometer, Shimadzu Corp.). Fluoresence measurements were carried out at 37°C. The ratio of the fluorescence intensities at the two excitation wavelengths was used to determine [Ca2+] i [21,22]. Calibration of [Ca2]i was by lysing the cells with 50 /zM digitonin in the presence of 1 mM CaC12 or 10 mM EGTA (pH 9.0) [23].
Other determinations Livers were removed and perfused with cold 0.15 M NaC1 to remove trapped blood. Identical lobes from each liver were ashed in a muffle furnace for 48 h at 450°C. The residue was then dissolved in 1.0 ml of concentrated HNO 3 (double distilled, GFS Chemicals, Columbus, OH) and returned to the muffle furnace for an additional 24 h. The remaining residue was dissolved in 2.0 ml of concentrated HC1 (reagent grade, Fisher Scientific, Fair Lawn, N J), diluted and analyzed
265 TABLE I
Indices o f copper status in rats fed copper deficient (Cu - ) or copper adequate (Cu + ) diets Liver and concentrations are based on the weight of the lyophilized liver sample. A unit of ceruloplasmin activity is that amount which catalyzes the oxidation of 1 p.mol o-dianisidine/min. Hematocrit and hemoglobin concentration were determined on whole blood using an electronic cell counter. Values are m e a n s + S.D.
Diet CuCu+
n Liver Cu (p.mol/kg)
Ceruloplasmin Hematocrit (units/l plasma) (fractional)
6 15.2+_ 4.3 a 0.4+- 0.6 a 6 150.7+-13.3 61.7_+32.7
Hemoglobin (g/l)
0.18+0.03 a 52_+11 a 0.42+-0.02 138+- 9
CuD
CuA
0 t~ LId 0 m LI.I
140nM-96nM
--
..~ n.-
70nM--
rV~l '
'
Thb
Thb
I
I
60s
Values for rats fed C u - are significantly lower than those for rats fed C u + ( P < 0.05, Student's t-test).
a
for and by flame atomic absorption spectroscopy [2]. Hematocrit, hemoglobin concentration and amine oxidase activity of plasma ceruloplasmin were determined as described in a previous report [2]. Data were analyzed statistically by analysis of variance (ANOVA) followed by Tukey's test for comparison between individual means or, when appropriate, by Student's t-test ( S A S / S T A T Version 6, SAS Institute, Cary, NC). Results
As shown in Table I, rats consuming diets containing low copper developed anemia and had significantly lower liver copper and plasma ceruloplasmin activity than their counterparts consuming diets containing adequate copper. These signs are characteristic of poor copper status [24-26] and confirm that the low copper diet induced copper deficiency in the rats consuming it. Fig. 1 shows examples of the change in [Ca2+] i that was induced by ionomycin treatment. Ionomycin (1
CuA
CuD
o
o lad .9 0 re"
Thb
10n0 I
I
60 s
?
t
Thb
Iono I
I
60s
Fig. 1. The release of internally stored Ca 2+ by ionomycin and subsequent stimulation of Ca 2÷ efflux by thrombin in platelets from copper-deficient (CUD) and control (CuA) rats. Platelets were suspended in the presence of 1.0 m M E G T A and stimulated with 1 p,M ionomycin (Iono) followed by 0.1 U / m l of thrombin (Thb). The ratio of fura-2 fluorescence signals at 340 n m and 380 n m excitation was used to calculate cytosolic Ca 2+ concentrations.
~
I
I
60s
Fig. 2. Changes in [Ca ~+ ]i following thrombin activation of platelets
from copper-deficient (CUD) and control (CuA) rats. Platelets were suspended in the presence of 1 mM EGTA and activated with 0.1 U/ml thrombin (Thb). The ratio of the fluorescence signals at 340 nm and 380 nm excitation was used to calculate cytosolic Ca 2+ concentrations. Fluorescenceemission was measured at 510 nm.
/xM), which in the presence of E G T A translocates internally stored Ca 2÷ to the cytosol [27,28], caused [Ca2+] i to rapidly increase in platelets from copper-deficient and control rats. In these particular examples, the maximum [Ca2+]i induced by ionomycin was less in platelets from the copper-deficient rat (152 nM) than in platelets from the control rat (210 nM). However, [Ca2÷]i in ionomycin-treated platelets from the copper-deficient rat remained elevated until the platelets were subsequently stimulated with thrombin (1 U / m l ) . In contrast, [Ca2+] i in ionomycin-treated platelets from the control rat declined after the initial rise. Generally, for all animals in the experiment, [Ca2+]i was essentially constant in ionomycin-treated platelets from copper-deficient rats, decreasing at a rate of only 3 + 3 n M / m i n , but steadily declined in ionomycin-treated platelets from control rats during the interval between peak [Ca2+]i and the addition of thrombin. After thrombin stimulation, the rate of [Ca2÷]i decline in platelets from copper-deficient rats (47_+ 17 n M / m i n ) became identical to the rate in platelets from control rats (42 + 15 n M / m i n ) . As shown in Fig. 2, thrombin (0.1 U / m l ) activation of platelets from copper-deficient and control rats caused a rapid rise i n [ C a 2 + ] i , which returned to resting concentrations within 60 s after stimulation. In these examples, the maximum [Ca2+] i induced by thrombin stimulation was lower in platelets from copper-deficient rats (96 nM) than in platelets from control rats (140 nM). The examples shown in Figs. 1 and 2 suggest that peak [Ca2+] i following either thrombin activation or ionomycin treatment may be lower in platelets from copper-deficient compared to control rats. Table II summarizes the effects of copper status, thrombin activation and ionomycin treatment on peak [Ca2+] i in
266 T A B L E II The effects o f dietary copper, thrombin activation and ionomycin treatment on platelet cytosolic Ca 2 + concentration The ratio of fura-2 fluorescence at 340 n m and 380 n m excitation was used to calculate [Ca 2+ li prior to and following treatment of platelets with 1 U / m l thrombin and 1 /xM ionomycin. [Ca 2+ ]i in stimulated platelets are peak concentrations. Values are m e a n s ± S.D. Diet
n
Cu Cu+
6 6
[Ca 2 + ]i (nM) resting
thrombin
Ionomycin
65 ± 16 77± 9
94_+ 19 a 131_+16
148 ± 27 ab 188±17b
a The stimulus, whether thrombin or ionomycin, produced lower [Ca2÷ ]i in platelets from rats fed C u - ( P < 0.05, Tukey's test). b Ionomycin treatment produced higher [Ca 2÷ ]i than thrombin activation regardless of dietary copper content ( P < 0.05, Tukey's test). Dietary copper did not significantly affect resting [Ca 2+]i ( P > 0.05, Tukey's test).
platelets from all copper-deficient and control rats. Analysis of these data by ANOVA indicated that changes in [Ca2+]i following treatment with either thrombin or ionomycin depended on copper status (treatment x status interaction, P < 0.05) and which activator was used (treatment × activator interaction, P < 0.05). Prior to thrombin stimulation, [ C a 2 + ] i w a s similar in platelets from copper-deficient and control rats. Following thrombin stimulation, peak [Ca2+] i was less in platelets from copper-deficient rats compared to platelets from control rats. P e a k [Ca2+]i also was less following ionomycin treatment of platelets from copper-deficient rats than platelets from control rats. Regardless of whether thrombin or ionomycin was used as an activator, peak [Ca2+] i was less in platelets from copper-deficient compared to control. Furthermore, peak [Ca 2+]i was higher in ionomycin-treated platelets
O
o,q. u.i o
80 n M- -
Thb
[ono
I
I
60s
Fig. 3. C h a n g e s in [Ca z+ ]i in platelets from a copper-deficient rat stimulated with 0.1 U / m l thrombin (Thb) followed by treatment with 1 g M ionomycin (Iono). T h e fluorescence ratio was used to calculate cytosolic Ca 2+ concentrations as in Figs. 1 and 2.
than in thrombin-activated platelets regardless of copper status. Because [Ca2+]i tended to remain elevated in ionomycin-treated platelets from copper-deficient rats until further treatment with thrombin (see Fig. 1), it was of interest to determine if [Ca2+] i would remain elevated following ionomycin treatment if the platelets were first activated by thrombin. As shown in Fig. 3, thrombin activation of platelets from a copper-deficient rat caused the usual rise and decay of [Ca2+] i. Additional treatment with ionomycin caused a second rise in [Ca2+] i which subsequently declined to the initial concentration. This result was typical of platelet samples from copper-deficient rats. Discussion
Past investigations have shown that deprivation of dietary copper alters cytoskeletal remodeling, accelerates dense granule secretion and may impair protein kinase C activation in thrombin-activated rat platelets [2,3]. Results from the present study suggest that copper deficiency also alters the thrombin-induced release of Ca 2+ from intracellular stores by reducing the size of these stores. In the presence of EGTA, treatment of platelets with 1 /~M ionomycin translocates internally stored Ca 2+ to the cytosol and prevents its resequestration into the storage pools [27,28]. Because ionomycin treatment maximally discharges the thrombin-sensitive Ca 2÷ pool [27], the size of this pool can be estimated from [Ca2+] i measured in platelets following ionomycin treatment [9]. In our study, peak [Ca2+] i in ionomycintreated platelets from control rats was 188 + 17 nM, which is similar to the previously reported value of 219 + 15 nM [9]. However, the peak lEa2+] i achieved following ionomycin treatment was significantly lowered by copper deficiency to 148 + 27 nM. Also, no further increases in [Ca2+] i occurred when ionomycintreated platelets from either copper-deficient or control rats were activated by thrombin, indicating that the thrombin-sensitive internal storage pools for Ca 2÷ were discharged by ionomycin and that no resequestration took place. Thus, it can be concluded that the amount of internally stored Ca 2+ in platelets is reduced by copper deficiency. Thrombin activation caused lEa2+] i to increase in platelets from copper-deficient and control rats. The peak [Ca 2÷ ]i following thrombin stimulation of platelets from control rats was 131 + 16 nM, which is comparable to the 161 + 6 nM previously reported for thrombin-activated rat platelets using similar experimental conditions [9]. However, the peak [Ca2+] i achieved following thrombin activation of platelets from copper-deficient rats was reduced to 94 + 20 nM. Because these experiments were performed with washed
267 platelets in the absence of extracellular Ca 2÷ and in the presence of E G T A , the increase in [Ca2+]i observed following thrombin activation of platelets from control and copper-deficient rats most likely represents release of Ca 2÷ from intracellular stores. However, as discussed above, copper deficiency reduces the size of the Ca 2÷ storage pool. Therefore, the rise in [Ca2+]i following thrombin activation of platelets from copper-deficient rats may be inhibited because less Ca 2÷ is available for release from internal stores. The decay of [Ca2+] i observed in ionomycin-treated platelets from control rats in the present study is consistent with previous results from ionomycin-treated human platelets [27-29]. Since the continued presence of ionomycin prevents resequestration of released Ca 2÷ back into the storage pools and does not contribute significantly to the efflux of Ca 2÷ from the cell, the decay of [Ca2+] i following ionomycin treatment in the presence of E G T A can be attributed to an active extrusion mechanism for Ca 2+ from the platelets [27,28]. After initially rising following ionomycin treatment, [Ca 2+]i remained essentially constant in platelets from copper-deficient rats. This implies that copper deficiency affects a mechanism or mechanisms responsible for transporting Ca 2+ out of platelets. However, thrombin activation subsequent to ionomycin treatment caused [Ca2+] i to decline at the same rate in platelets from copper-deficient and control rats. These observations indicate that copper deficiency may impair a constitutive, receptor-independent mechanism for the extrusion of Ca 2+ from platelets but has little effect on receptor-operated mechanisms for Ca 2+ efflux. Furthermore, once platelets from copper-deficient rats were activated by thrombin, their ability to remove Ca 2÷ released into the cytosol from internal stores by ionomycin treatment was not affected. This suggests that copper deficiency has little effect on the processing of Ca 2+ mobilized from internal stores once thrombin activation has taken place. It is not possible to address the nature of the Ca 2÷ transport mechanisms in rat platelets using results from the present study. However, human platelets contain at least two biochemically distinct Ca2+-ATPases in the plasma and intracellular membranes [30,31]. It is possible, therefore, that rat platelets also contain multiple Ca2+-ATPases, some of which are not receptor operated but provide a means for Ca 2+ transport into intracellular stores or out of the cell to help maintain Ca 2+ homeostasis. Inactivation of such a Ca2+-ATPase could explain the lower Ca 2+ stores in platelets from copper-deficient rats and also the inhibition of Ca 2+ efflux from these cells once the intracellular stores are discharged with ionomycin. The findings of the present study indicate that copper deficiency reduces the peak [Ca2+]i in platelets following thrombin activation but does not impair re-
ceptor-operated mechanisms for lowering [Ca2+]i once Ca 2+ is mobilized from internal stores. However, increased or sustained [Ca2+]i would be required to directly enhance thrombin-induced actin polymerization and myosin-actin interaction. Thus, the changes caused by copper deficiency on Ca 2+ mobilization from internal stores, which tends to lower [Ca2+]i in the absence of extracellular Ca 2÷, cannot explain the increase in actin polymerization and myosin association with actin that occurs in thrombin-stimulated platelets during copper deficiency. However, lower [Ca2+]i may impair Ca2+-activated mechanisms that negatively control these platelet responses, leading to their increase during copper deficiency.
Acknowledgements The authors would like to thank Pam Sakkinen for technical assistance and Lu Ann Johnson for performing statistical analysis of our data.
References 1 Holmsen, H. (1989) Ann. Med. 21, 23-30. 2 Johnson, W.T. and Dufault, S.N. (1989) J. Nutr. 119, 1404-1410. 3 Johnson, W.T. and Dufault, S.N. (1991) J. Nutr. Biochem. 2, 663-670. 4 Siess, W. (1989) Physiol. Rev. 69, 58-178. 5 Zucker, M.B. and Nachmias, V.T. (1985) Arteriosclerosis 5, 2-18. 6 Rink, T.J., Smith, S.W. and Tsein, R.Y. (1982) FEBS Lett. 148, 21-26. 7 Wenche, J. and Haynes, D.H. (1987) Biochim. Biophys. Acta 929, 88-102. 8 Thompson, N.T. and Scrutton, M.C. (1985) Eur. J. Biochem. 147, 421-427. 9 Heemslerk, J.W.M., Feijge, M.A.H., Reitman, E. and Hornstra, G. (1991) FEBS Len. 284, 223-226. 10 Fox, J.E.B. and Phillips, D.R. (1983) Semin. Hematol. 20, 243260. 11 Stossel, T.P. (1989) J. Biol. Chem. 264, 18261-18264. 12 Janmey, P.A., Chaponnier, C., Lind, S.E., Zaner, K.S., Stossel, T.P. and Yin, Y.L. (1985) Biochemistry 24, 3714-3723. 13 Fox, J.E.B. and Phillips, D.R. (1982) J. Biol. Chem. 257, 41204126. 14 Nishizuka, Y. (1984) Nature 308, 693-698. 15 Bell, R.M. (1986) Cell 45, 631-632. 16 Nishikawa, M., Tanaka, T. and Hidaka, H. (1980) Nature 287, 863-865. 17 Daniel, J.L., Holmsen, H. and Adelstein, R.S. (1977) Thrombos. Haemostas. 38, 984-989. 18 Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fijikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704. 19 Johnson, W.T. and Kramer, T.R. (1987) J. Nutr. 117, 1085-1090. 20 Jennings, L.K., Fox, J.E.B., Edwards, H.H. and Phillips, D.R. (1981) J. Biol. Chem. 256, 6927-6932. 21 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. 22 Cobbold, P.H. and Rink, T.J. (1987) Biochem. J. 248, 313-328. 23 Schaeffer,J. and Blaustein, M.P. (1989) Cell Calcium 10, 101-113. 24 Owen, C.A., Jr. and Hazelrig, J.B. (1968) Am. J. Physiol. 215, 334-338. 25 Alfaro, B. and Heaton, F.W. (1973) Br. J. Nutr. 29, 73-85.
268 26 Evans, J.L. and Abraham, P.A. (1973) J. Nutr. 103, 196-201. 27 Pollock, W.K., Sage, S.O, and Rink, T.J. (1987) FEBS Lett. 2, 132-136. 28 Johansson, J.S. and Haynes, D.H. (1988) J. Membrane Biol. 104, 147-163.
29 Rink, T.R. and Sage, S.O. (1987) J. Physiol. 393, 513-524. 30 Enouf, J., Bredoux, R., Bourdeau, N. and Levy-Toledano, S. (1987) J. Biol. Chem, 262, 9293-9297. 31 Enouf, J., Bredoux, R., Bourdeau, N., Sarkadi, B. and LevyToledano, S. (1989) Biochem. J. 263, 547-552.