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Effects of Insulin and Insulin-like Growth Factor-1 on Intracellular Magnesium of Platelets
EXPERIMENTAL AND MOLECULAR PATHOLOGY ARTICLE NO.
65, 104–109 (1998)
MO982232
Effects of Insulin and Insulin-like Growth Factor-1 on Intracellular Magnesium of Platelets Junji Takaya, Hirohiko Higashino, Rika Miyazaki, and Yohnosuke Kobayashi Department of Pediatrics, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8506, Japan Received March 2, 1998, and in revised form August 3, 1998 Magnesium (Mg2+), the second most abundant intracellular cation, is a critical cofactor in numerous enzymatic reactions. After stimulation of platelets with insulin and insulin-like growth factor-1 (IGF-1), we examined changes in cytosolic free Mg2+ ([Mg2+]i) by using fluorescent probe magfura2. Basal [Mg2+]i in platelets was 614 6 18 mM (mean 6 SEM, n 5 60). Insulin and IGF-1 induced an immediate rise of [Mg2+]i in a dose-dependent manner. After stimulation of platelets with 100 mU/mL of insulin for 60 s, [Mg2+]i was significantly elevated to 1270 6 53 mM (n 5 30, P , 0.05), i.e., 82 6 5% over resting [Mg2+]i. IGF-1 (5 mg/mL) also increased [Mg2+]i (1020 6 53 mM, 69 6 10% over resting [Mg2+]i, n 5 30). In the medium containing choline instead of sodium or the medium without potassium, an elevation of [Mg2+]i with addition of insulin/IGF-1 was moderately suppressed. Amiloride, a Na+-H+ antiport inhibitor, did not block the insulin/IGF-1 effect. Insulin/ IGF-1 translocates Mg2+ from the extracellular space to intracellular space and these effects are affected by external sodium and potassium. q 1998 Academic Press
INTRODUCTION Magnesium, the second most abundant intracellular cation, is an essential factor in the glucose transport mechanism across the cell membranes (Paolisso et al., 1990). It is also a critical cofactor in numerous enzymatic reactions (Flatman, 1984). Under the experimental condition, its intracellular level is precisely regulated within a narrow range in spite of a wide variation in external Mg2+ concentration (Flatman, 1991). This implies the existence of a specialized Mg2+ transport system. An early event in the agonist action involves the translocation of ions, and the translocation of critical ions in turn is required to activate one or more components of the cellular synthetic machinery (Flatman, 1991). A variety of growth factors act on quiescent mammalian cells leading to an increase in the fluxes of Na+, K+, and H+ across the plasma membrane and to stimulation of Ca2+ mobilization (Rozengurt and Mendoza, 1986). There is little documentation on the mobilization of Mg2+ in response to growth factors. Receptors for IGF-1 and insulin have been demonstrated on human platelets (Hartmann et al., 1989; Trovati et al., 1988). However, factors or hormones regulating Mg2+ transport and its relation to platelet activity remain unclear. Our purpose is to clarify what factors would influence the intracellular Mg2+. MATERIALS AND METHODS Platelet Preparation Human platelets were obtained from healthy volunteer donors and were isolated as previously described (Takaya et al., 1997). Approximately 10 ml of venous blood was drawn into 3.8% (w/v) acid citrate buffer (10:1, v/v) and was centrifuged at 200g for 10 min at room temperature. The platelet-rich plasma was decanted and further centrifuged 104 0014-4800/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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at 1000g for 10 min, and the cells were washed three times in Hepes buffer solution (HBS) containing (in mM): NaCl, 140; KCl, 5; glucose, 25; MgCl2, 1; NaH2PO4, 1; Hepes, 25 (pH 7.2); and EGTA, 0.2. EGTA was omitted from the third washing, and 0.1% fatty-acid-free bovine serum albumin (BSA) was added. Platelets were counted in a Celltac counter (Nihon Kohden, Tokyo, Japan). Unless otherwise indicated, platelets were suspended in HBS at a concentration of (2–3) 3 107 platelets/ml. Platelets were studied within 4 h after blood drawing. Measurements of Intracellular Calcium and Magnesium Concentrations Intracellular ionic calcium ([Ca2+]i) and [Mg2+]i concentrations were measured with a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Instruments, Tokyo, Japan) by using Ca-specific or Mg-specific fura-2 probes as described by Grynkiewicz et al. (Grynkiewicz et al., 1985; Raju et al., 1989). Two micromolar Ca-fura-2/acetoxymethyl (for [Ca2+]i measurement) or mag-fura-2/acetoxymethyl (for [Mg2+]i measurement) dye was added to the platelet suspension and incubated at 378C for 30 min. After loading of the dyes, the platelets were washed twice with HBS, the fura dyes were removed by centrifugation, and the platelets were resuspended in HBS. In each [Ca2+]i measurement, 1 mM CaCl2 was present in the medium. The excitation wavelengths were set at 340/380 nm (for [Ca2+]i) or 335/370 nm (for [Mg2+]i), and the emission wavelength was 510 nm for both. Each intracellular ionic concentration was calculated as described (Grynkiewicz et al., 1985; Raju et al., 1989) by using dissociation constant (Kd) 5 224 (nM) for Ca and Kd 5 1500 (mM) for Mg. The maximum intensities were determined by disrupting the cells with 0.1% Triton in the presence of 30 mM MgCl2 (for [Mg2+]i) or 10 mM CaCl2 (for [Ca2+]i). The minimum intensities were the values determined in the presence of 60 mM EDTA (for [Mg2+]i) or 20 mM ethyleneglycol-bis(b-aminoethyl ether)-N,N8-tetraacetic acid (for [Ca2+]i). MnCl2 (0.05 mM) was used to quench the fluorescence from extracellular dye according to the methods of Ng et al. (1991). Insulin, IGF-1, and tetraethylammonium chloride were dissolved in deionized water. Twenty-five microliters of insulin, IGF-1, and each agonist was added to 2.5 ml of platelet suspension. Chemicals All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Pertussis toxin was from Research Biochemicals International (Natick, MA). Tetraethylammonium chloride and glibenclamide was from Wako (Osaka, Japan). Cafura-2/acetoxymethyl was from Dojin (Kumamoto, Japan) and mag-fura-2/acetoxymethyl was from Molecular Probes (Eugene, OR). IGF-1 was a gift from Fujisawa Pharmaceutical Co. Ltd. (Tokyo, Japan). Statistical Analysis Results of the experiments were expressed as the means 6 SEM. Statistical significance was assessed by Student’s t test. For intergroup comparisons, data were subjected to oneway ANOVA. A P value of ,0.05 was considered to be of statistical significance. RESULTS Basal [Mg2+]i in platelets was 614 6 18 mM (n 5 60) at an external magnesium concentration of 1 mM. Basal[Ca2+]i in platelets was 78 6 8 nM (n 5 21). The dose– response effect of insulin on the changes of [Mg2+]i was measured in washed platelets
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suspended in HBS. Insulin, added additionally, increased [Mg2+]i of mag-fura-2-loaded platelets in a dose-dependent manner (Fig. 1, top). IGF-1 also increased [Mg2+]i; the maximal extent of the increase was, however, smaller than that observed with insulin (Fig. 1, bottom). After 60 s of stimulation with 100 mU/mL insulin or 5 mg/ml IGF-1, [Mg2+]i was significantly elevated to 1270 6 53 mM (n 5 30, 82 6 5%) and 1020 6 53 mM (n 5 30, 68 6 11%), respectively, over resting [Mg2+]i. When platelets were treated with 3 mM EGTA, the insulin/IGF-1 effect on [Mg2+]i increase was not observed. In a medium containing 140 mM choline instead of Na+, an increase in [Mg2+]i with addition of insulin/IGF-1 was reduced (n 5 4; 222.4 6 4 and 241.5 6 5%, respectively). The increase in [Mg2+]i due to insulin/IGF-1 was reduced in a medium without potassium (n 5 4; 249 6 7 and 241 6 19%, respectively). Insulin/IGF-1 alone had no effect on the [Ca2+]i in the presence of 1 mM Ca2+. In the presence of 25 mM glucose, additional glucose (28 mM) increased [Mg2+]i (n 5 15, 849 6 92 mM, 39 6 6% over resting [Mg2+]i). However, additional glucose was not effective in altering [Ca2+]i in the presence of 1 mM CaCl2 (data not shown). Thrombin (0.1 NIH Units/ml) increased [Ca2+]i in the medium containing 1 mM CaCl2 (n 5 20, 370 6 25 nM, 505 6 88% over resting [Ca2+]i). However, thrombin decreased [Mg2+]i
FIG. 1. Time course of insulin and IGF-1 effects on platelet free magnesium level. Dose–response effects of insulin (top) and IGF-1 (bottom) on cytoplasmic free Mg2+ concentration ([Mg2+]i) of mag-fura-2-loaded platelets. Arrows indicate successive additions of insulin and IGF-1. After each addition of insulin and IGF-1, the concentrations were 1, 11, 61, 161, and 361 mU/ml and 0.05, 0.55, and 5.55 mg/ml, respectively.
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FIG. 2. Time course of thrombin effect on magnesium transport in platelets. Dose–response effects of thrombin on cytoplasmic free Mg2+ concentration ([Mg2+]i) of mag-fura-2-loaded platelets. Arrows indicate additions of thrombin. After each addition of thrombin, the concentrations were 0.025, 0.075, 0.175, and 0.375 NIHU/ml.
in a dose-dependent manner (Fig. 2) in the medium without 1 mM CaCl2. Thrombin (0.1 NIH Units/ml) and 1 mM ATP (Fig. 3) decreased [Mg2+]i (n 5 20, 234 6 3%; and n 5 10, 253 6 7% than resting [Mg2+]i, respectively). ADP (0.1 mM), GTP (0.1 mM), GDP (1 mM), and 8-bromo-cyclic guanosine monophosphate (0.1 mM) and 8-bromo-adenosine
FIG. 3. Time course of agonist-stimulated effect on magnesium transport in platelets. (Top) Representative tracings of 0.1 mM ADP, 0.1 mM GTP, 1 mM GDP, and 1 mM ATP-induced changes in [Mg2+]i. (Bottom) Representative tracings of 0.1 mM 8Br-cGMP, 0.1 mM 8Br-cAMP, 1 mM ATP, and 100 mU/mL insulin-induced changes in [Mg2+]i.
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38-58 cyclic monophosphate (0.1 mM) neither changed [Mg2+]i nor blocked the effect of insulin/IGF-1. Separate additions of nucleotides (0.1–1.0 mM) other than ATP did not induce any change in [Mg2+]i level (data not shown). Pretreatment of platelets with pertussis toxin (1 mg/ml) had no effect on the insulin/ IGF-1 reaction (data not shown). Ouabain (0.1 mM), which inhibits (Na+-K+) ATPase, did not block the increase in [Mg2+]i due to insulin/IGF-1. Amiloride (0.5–2.0 mM), which inhibits Na+-H+ antiport, also did not block the insulin/IGF-1 effect. Tetraethylammonium chloride (0.1 mM) and glibenclamide (15 mM), which inhibit the potassium channel, did not block the insulin/IGF-1 effects. These suspensions with inhibitors were incubated at 378C for 30 min before the addition of insulin/IGF-1 and [Mg2+]i measurement. DISCUSSION Insulin and IGF-1 induced an immediate rise of [Mg2+]i in platelets. No increase in [Mg2+]i was observed when extracellular Mg2+ was chelated. This observation is interpreted to indicate that insulin/IGF-1 translocate Mg2+ from the extracellular to intracellular space. Insulin/IGF-1 may hyperpolarize platelets by increasing Mg2+ influx through voltageoperated channels. Mg2+ is generally considered unlikely to act as a second messenger, because [Mg2+]i is reported not to change dramatically like [Ca2+]i (Flatman, 1984, 1991). In our study insulin induced a more rapid increase in [Mg2+]i than has been reported previously (Hwang et al., 1993) with the same insulin dose. The increase in [Mg2+]i was barely observed at a physiological insulin or IGF-1 concentration (normal concentrations: 5–20 mU/ml and 180–780 ng/ml, respectively) (Underwood and Van Wyk, 1992). Our observations suggest that [Mg2+]i may act as a messenger because [Mg2+]i instantaneously changes following stimulation with various biologically active substances. The values for basal [Mg2+]i were high at 614 6 18 mM. Other investigators reported values of 266 6 28 mM (SD) (Hwang et al., 1993) and 421 6 52 mM (SEM) (Yoshimura et al., 1995). Although there is a wide range of the resting values of [Mg2+]i, the differences in medium and wavelengths may account for such variation. The source of energy for Mg2+ transport may be the coupling of Mg2+ entry to the obligatory exit of either Na+ or K+, which travels down its electrochemical gradient. Our study indicates that the rapid translocation of Mg2+ with insulin/IGF-1 may be independent of the potassium channel, which is inhibited by tetraethylammonium chloride and glibenclamide. It was reported that some channels are affected by nucleotides or cyclic nucleotides (Lee and Laychock, 1997; Yokoshiki et al., 1998). Our results showed that only ATP had effects on [Mg2+]i. The relationship between [Mg2+]i and thrombin stimulated [Ca2+]i and exact mechanisms whereby thrombin regulates [Mg2+]i still remain to be elucidated. As EGTA has more affinity for Ca2+ than for Mg2+ (Grynkiewicz et al., 1985), removal of Ca2+ may have had a more potent effect on platelet function than chelation of some Mg2+. We studied another pathway of Mg2+ transport. Several pumps and transporters are present in platelets (Rink and Sage, 1990; Kimura and Aviv, 1993). In muscle, insulin/ IGF-1 stimulates the Na+-K+ pump (Dørup and Clausen, 1995). The accumulation of Mg2+ caused by insulin/IGF-1 can occur even if the Mg2+-requiring (Na+-K+) ATPase is impaired by ouabain. Mg2+ efflux depends on the extent of the Na+ gradient across the membrane. Wasserman et al. reported that Na+/Mg2+ exchange can also occur in the reverse direction (Wasserman et al., 1986). Yoshimura et al. reported that Na+/Mg2+ exchange might be involved in the regulation of [Mg2+]i in human platelets (Yoshimura et al., 1995). Amiloride, an inhibitor of Na+-H+ antiport, did not block the increasing effect of insulin/IGF-1 on [Mg2+]i. These data suggest that Mg2+ influx with insulin/IGF-1 from extracellular
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+
2+
space is independent of Na -H antiport. The influx pathway for [Mg ]i by insulin/IGF1 is uncertain. Insulin/IGF-1 translocate Mg2+ from the extracellular space to intracellular space and these effects are affected by external sodium and potassium. ACKNOWLEDGMENTS This study was supported by Research Grants from the Morinaga Hoshikai and the Mami Mizutani Foundations.
REFERENCES Dørup, I., and Clausen, T. (1995). Insulin-like growth factor I stimulates active Na+-K+ transport in rat soleus muscle. Am. J. Physiol. 268, E849–E857. Flatman, P. W. (1991). Mechanisms of magnesium transport. Annu. Rev. Physiol. 53, 259–271. Flatman, P. W. (1984). Magnesium transport across cell membranes. J. Membr. Biol. 80, 1–14. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Hartmann, K., Baier, T. G., Loibl, R., Schmitt, A., and Schonberg, D. (1989). Demonstration of type I insulinlike growth factor receptors on human platelets. J. Recept. Res. 9, 181–198. Hwang, D. L., Yen, C. F., and Nadler, J. L. (1993). Insulin increases intracellular magnesium transport in human platelets. J. Clin. Endocrinol. Metab. 76, 549–553. Kimura, M., and Aviv, A. (1993). Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: The roles of the Na+/Ca2+ exchange, the Na+-K+-2Cl2 cotransport and cellular volume. Pflugers Arch. 422, 585–590. Lee, B., and Laychock, S. G. (1997). Atrial natriuretic peptide and cyclic nucleotides affect glucose-induced Ca2+ responses in single pancreatic islet b-cells. Diabetes 46, 1312–1318. Ng, L. L., Davies, J. E., and Garrido, M. C. (1991). Intracellular free magnesium in human lymphocytes and the response to lectins. Clin. Sci. 80, 539–547. ` Paolisso, G., Scheen, A., D’Onofrio, F., and Lefebvre, P. (1990). Magnesium and glucose homeostasis. Diabetologia 33, 511–514. Raju, B., Murphy, E., Levy, L. A., Hall, R. D., and London, R. E. (1989). A fluorescent indicator for measuring cytosolic free magnesium. Am. J. Physiol. 256, C540–C548. Rink, T. J., and Sage, S. O. (1990). Calcium signaling in human platelets. Annu. Rev. Physiol. 52, 431–449. Rozengurt, E., and Mendoza, S. A. (1986). Early stimulation of Na+-H+ antiport, Na+-K+ pump activity and Ca2+ fluxes in fibroblast mitogenesis. Curr. Top. Membr. Transp. 27, 163–191. Schachter, M., Gallagher, K. L., and Sever, P. S. (1990). Measurement of intracellular magnesium in a vascular smooth muscle cell line using a fluorescent probe. Biochem. Biophys. Acta 1035, 378–380. Takaya, J., Iwamoto, Y., Higashino, H., Ishihara, R., and Kobayashi, Y. (1997). Increased intracellular calcium and altered phorbol dibutyrate binding to intact platelets in young subjects with insulin-dependent and noninsulin-dependent diabetes mellitus. Metabolism 46, 949–953. Trovati, M., Anfossi, G., Cavalot, F., Massucco, P., and Emanuelli, G. (1988). Insulin directly reduces platelet sensitivity to aggregating agents. Diabetes 37, 780–786. Underwood, L. E., and Van Wyk, J. J. (1992). Normal and aberrant growth. In “Williams Textbook of Endocrinology” (J. D. Wilson and D. W. Foster, Eds.), 8th ed., pp. 1079–1138. Saunders, Philadelphia. Wasserman, A., McClellan, G., and Somlyo, A. P. (1986). Calcium-sensitive cellular and subcellular transport of sodium, potassium, magnesium, and calcium in sodium-loaded vascular smooth muscle. Circ. Res. 58, 790–802. Yokoshiki, H., Sunabawa, M., Seki, T., and Sperelakis, N. (1998). ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am. J. Physiol. 274, C25–C37. Yoshimura, M., Oshima, T., Matsuura, H., Watanabe, M., Higashi, Y., Ono, N., Hiraga, H., Kambe, M., and Kajiyama, G. (1995). Effect of the transmembrane gradient of magnesium and sodium on the regulation of cytosolic free magnesium concentration in human platelets. Clin. Sci. 89, 293–298.
65, 104–109 (1998)
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Effects of Insulin and Insulin-like Growth Factor-1 on Intracellular Magnesium of Platelets Junji Takaya, Hirohiko Higashino, Rika Miyazaki, and Yohnosuke Kobayashi Department of Pediatrics, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8506, Japan Received March 2, 1998, and in revised form August 3, 1998 Magnesium (Mg2+), the second most abundant intracellular cation, is a critical cofactor in numerous enzymatic reactions. After stimulation of platelets with insulin and insulin-like growth factor-1 (IGF-1), we examined changes in cytosolic free Mg2+ ([Mg2+]i) by using fluorescent probe magfura2. Basal [Mg2+]i in platelets was 614 6 18 mM (mean 6 SEM, n 5 60). Insulin and IGF-1 induced an immediate rise of [Mg2+]i in a dose-dependent manner. After stimulation of platelets with 100 mU/mL of insulin for 60 s, [Mg2+]i was significantly elevated to 1270 6 53 mM (n 5 30, P , 0.05), i.e., 82 6 5% over resting [Mg2+]i. IGF-1 (5 mg/mL) also increased [Mg2+]i (1020 6 53 mM, 69 6 10% over resting [Mg2+]i, n 5 30). In the medium containing choline instead of sodium or the medium without potassium, an elevation of [Mg2+]i with addition of insulin/IGF-1 was moderately suppressed. Amiloride, a Na+-H+ antiport inhibitor, did not block the insulin/IGF-1 effect. Insulin/ IGF-1 translocates Mg2+ from the extracellular space to intracellular space and these effects are affected by external sodium and potassium. q 1998 Academic Press
INTRODUCTION Magnesium, the second most abundant intracellular cation, is an essential factor in the glucose transport mechanism across the cell membranes (Paolisso et al., 1990). It is also a critical cofactor in numerous enzymatic reactions (Flatman, 1984). Under the experimental condition, its intracellular level is precisely regulated within a narrow range in spite of a wide variation in external Mg2+ concentration (Flatman, 1991). This implies the existence of a specialized Mg2+ transport system. An early event in the agonist action involves the translocation of ions, and the translocation of critical ions in turn is required to activate one or more components of the cellular synthetic machinery (Flatman, 1991). A variety of growth factors act on quiescent mammalian cells leading to an increase in the fluxes of Na+, K+, and H+ across the plasma membrane and to stimulation of Ca2+ mobilization (Rozengurt and Mendoza, 1986). There is little documentation on the mobilization of Mg2+ in response to growth factors. Receptors for IGF-1 and insulin have been demonstrated on human platelets (Hartmann et al., 1989; Trovati et al., 1988). However, factors or hormones regulating Mg2+ transport and its relation to platelet activity remain unclear. Our purpose is to clarify what factors would influence the intracellular Mg2+. MATERIALS AND METHODS Platelet Preparation Human platelets were obtained from healthy volunteer donors and were isolated as previously described (Takaya et al., 1997). Approximately 10 ml of venous blood was drawn into 3.8% (w/v) acid citrate buffer (10:1, v/v) and was centrifuged at 200g for 10 min at room temperature. The platelet-rich plasma was decanted and further centrifuged 104 0014-4800/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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at 1000g for 10 min, and the cells were washed three times in Hepes buffer solution (HBS) containing (in mM): NaCl, 140; KCl, 5; glucose, 25; MgCl2, 1; NaH2PO4, 1; Hepes, 25 (pH 7.2); and EGTA, 0.2. EGTA was omitted from the third washing, and 0.1% fatty-acid-free bovine serum albumin (BSA) was added. Platelets were counted in a Celltac counter (Nihon Kohden, Tokyo, Japan). Unless otherwise indicated, platelets were suspended in HBS at a concentration of (2–3) 3 107 platelets/ml. Platelets were studied within 4 h after blood drawing. Measurements of Intracellular Calcium and Magnesium Concentrations Intracellular ionic calcium ([Ca2+]i) and [Mg2+]i concentrations were measured with a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Instruments, Tokyo, Japan) by using Ca-specific or Mg-specific fura-2 probes as described by Grynkiewicz et al. (Grynkiewicz et al., 1985; Raju et al., 1989). Two micromolar Ca-fura-2/acetoxymethyl (for [Ca2+]i measurement) or mag-fura-2/acetoxymethyl (for [Mg2+]i measurement) dye was added to the platelet suspension and incubated at 378C for 30 min. After loading of the dyes, the platelets were washed twice with HBS, the fura dyes were removed by centrifugation, and the platelets were resuspended in HBS. In each [Ca2+]i measurement, 1 mM CaCl2 was present in the medium. The excitation wavelengths were set at 340/380 nm (for [Ca2+]i) or 335/370 nm (for [Mg2+]i), and the emission wavelength was 510 nm for both. Each intracellular ionic concentration was calculated as described (Grynkiewicz et al., 1985; Raju et al., 1989) by using dissociation constant (Kd) 5 224 (nM) for Ca and Kd 5 1500 (mM) for Mg. The maximum intensities were determined by disrupting the cells with 0.1% Triton in the presence of 30 mM MgCl2 (for [Mg2+]i) or 10 mM CaCl2 (for [Ca2+]i). The minimum intensities were the values determined in the presence of 60 mM EDTA (for [Mg2+]i) or 20 mM ethyleneglycol-bis(b-aminoethyl ether)-N,N8-tetraacetic acid (for [Ca2+]i). MnCl2 (0.05 mM) was used to quench the fluorescence from extracellular dye according to the methods of Ng et al. (1991). Insulin, IGF-1, and tetraethylammonium chloride were dissolved in deionized water. Twenty-five microliters of insulin, IGF-1, and each agonist was added to 2.5 ml of platelet suspension. Chemicals All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Pertussis toxin was from Research Biochemicals International (Natick, MA). Tetraethylammonium chloride and glibenclamide was from Wako (Osaka, Japan). Cafura-2/acetoxymethyl was from Dojin (Kumamoto, Japan) and mag-fura-2/acetoxymethyl was from Molecular Probes (Eugene, OR). IGF-1 was a gift from Fujisawa Pharmaceutical Co. Ltd. (Tokyo, Japan). Statistical Analysis Results of the experiments were expressed as the means 6 SEM. Statistical significance was assessed by Student’s t test. For intergroup comparisons, data were subjected to oneway ANOVA. A P value of ,0.05 was considered to be of statistical significance. RESULTS Basal [Mg2+]i in platelets was 614 6 18 mM (n 5 60) at an external magnesium concentration of 1 mM. Basal[Ca2+]i in platelets was 78 6 8 nM (n 5 21). The dose– response effect of insulin on the changes of [Mg2+]i was measured in washed platelets
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suspended in HBS. Insulin, added additionally, increased [Mg2+]i of mag-fura-2-loaded platelets in a dose-dependent manner (Fig. 1, top). IGF-1 also increased [Mg2+]i; the maximal extent of the increase was, however, smaller than that observed with insulin (Fig. 1, bottom). After 60 s of stimulation with 100 mU/mL insulin or 5 mg/ml IGF-1, [Mg2+]i was significantly elevated to 1270 6 53 mM (n 5 30, 82 6 5%) and 1020 6 53 mM (n 5 30, 68 6 11%), respectively, over resting [Mg2+]i. When platelets were treated with 3 mM EGTA, the insulin/IGF-1 effect on [Mg2+]i increase was not observed. In a medium containing 140 mM choline instead of Na+, an increase in [Mg2+]i with addition of insulin/IGF-1 was reduced (n 5 4; 222.4 6 4 and 241.5 6 5%, respectively). The increase in [Mg2+]i due to insulin/IGF-1 was reduced in a medium without potassium (n 5 4; 249 6 7 and 241 6 19%, respectively). Insulin/IGF-1 alone had no effect on the [Ca2+]i in the presence of 1 mM Ca2+. In the presence of 25 mM glucose, additional glucose (28 mM) increased [Mg2+]i (n 5 15, 849 6 92 mM, 39 6 6% over resting [Mg2+]i). However, additional glucose was not effective in altering [Ca2+]i in the presence of 1 mM CaCl2 (data not shown). Thrombin (0.1 NIH Units/ml) increased [Ca2+]i in the medium containing 1 mM CaCl2 (n 5 20, 370 6 25 nM, 505 6 88% over resting [Ca2+]i). However, thrombin decreased [Mg2+]i
FIG. 1. Time course of insulin and IGF-1 effects on platelet free magnesium level. Dose–response effects of insulin (top) and IGF-1 (bottom) on cytoplasmic free Mg2+ concentration ([Mg2+]i) of mag-fura-2-loaded platelets. Arrows indicate successive additions of insulin and IGF-1. After each addition of insulin and IGF-1, the concentrations were 1, 11, 61, 161, and 361 mU/ml and 0.05, 0.55, and 5.55 mg/ml, respectively.
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FIG. 2. Time course of thrombin effect on magnesium transport in platelets. Dose–response effects of thrombin on cytoplasmic free Mg2+ concentration ([Mg2+]i) of mag-fura-2-loaded platelets. Arrows indicate additions of thrombin. After each addition of thrombin, the concentrations were 0.025, 0.075, 0.175, and 0.375 NIHU/ml.
in a dose-dependent manner (Fig. 2) in the medium without 1 mM CaCl2. Thrombin (0.1 NIH Units/ml) and 1 mM ATP (Fig. 3) decreased [Mg2+]i (n 5 20, 234 6 3%; and n 5 10, 253 6 7% than resting [Mg2+]i, respectively). ADP (0.1 mM), GTP (0.1 mM), GDP (1 mM), and 8-bromo-cyclic guanosine monophosphate (0.1 mM) and 8-bromo-adenosine
FIG. 3. Time course of agonist-stimulated effect on magnesium transport in platelets. (Top) Representative tracings of 0.1 mM ADP, 0.1 mM GTP, 1 mM GDP, and 1 mM ATP-induced changes in [Mg2+]i. (Bottom) Representative tracings of 0.1 mM 8Br-cGMP, 0.1 mM 8Br-cAMP, 1 mM ATP, and 100 mU/mL insulin-induced changes in [Mg2+]i.
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38-58 cyclic monophosphate (0.1 mM) neither changed [Mg2+]i nor blocked the effect of insulin/IGF-1. Separate additions of nucleotides (0.1–1.0 mM) other than ATP did not induce any change in [Mg2+]i level (data not shown). Pretreatment of platelets with pertussis toxin (1 mg/ml) had no effect on the insulin/ IGF-1 reaction (data not shown). Ouabain (0.1 mM), which inhibits (Na+-K+) ATPase, did not block the increase in [Mg2+]i due to insulin/IGF-1. Amiloride (0.5–2.0 mM), which inhibits Na+-H+ antiport, also did not block the insulin/IGF-1 effect. Tetraethylammonium chloride (0.1 mM) and glibenclamide (15 mM), which inhibit the potassium channel, did not block the insulin/IGF-1 effects. These suspensions with inhibitors were incubated at 378C for 30 min before the addition of insulin/IGF-1 and [Mg2+]i measurement. DISCUSSION Insulin and IGF-1 induced an immediate rise of [Mg2+]i in platelets. No increase in [Mg2+]i was observed when extracellular Mg2+ was chelated. This observation is interpreted to indicate that insulin/IGF-1 translocate Mg2+ from the extracellular to intracellular space. Insulin/IGF-1 may hyperpolarize platelets by increasing Mg2+ influx through voltageoperated channels. Mg2+ is generally considered unlikely to act as a second messenger, because [Mg2+]i is reported not to change dramatically like [Ca2+]i (Flatman, 1984, 1991). In our study insulin induced a more rapid increase in [Mg2+]i than has been reported previously (Hwang et al., 1993) with the same insulin dose. The increase in [Mg2+]i was barely observed at a physiological insulin or IGF-1 concentration (normal concentrations: 5–20 mU/ml and 180–780 ng/ml, respectively) (Underwood and Van Wyk, 1992). Our observations suggest that [Mg2+]i may act as a messenger because [Mg2+]i instantaneously changes following stimulation with various biologically active substances. The values for basal [Mg2+]i were high at 614 6 18 mM. Other investigators reported values of 266 6 28 mM (SD) (Hwang et al., 1993) and 421 6 52 mM (SEM) (Yoshimura et al., 1995). Although there is a wide range of the resting values of [Mg2+]i, the differences in medium and wavelengths may account for such variation. The source of energy for Mg2+ transport may be the coupling of Mg2+ entry to the obligatory exit of either Na+ or K+, which travels down its electrochemical gradient. Our study indicates that the rapid translocation of Mg2+ with insulin/IGF-1 may be independent of the potassium channel, which is inhibited by tetraethylammonium chloride and glibenclamide. It was reported that some channels are affected by nucleotides or cyclic nucleotides (Lee and Laychock, 1997; Yokoshiki et al., 1998). Our results showed that only ATP had effects on [Mg2+]i. The relationship between [Mg2+]i and thrombin stimulated [Ca2+]i and exact mechanisms whereby thrombin regulates [Mg2+]i still remain to be elucidated. As EGTA has more affinity for Ca2+ than for Mg2+ (Grynkiewicz et al., 1985), removal of Ca2+ may have had a more potent effect on platelet function than chelation of some Mg2+. We studied another pathway of Mg2+ transport. Several pumps and transporters are present in platelets (Rink and Sage, 1990; Kimura and Aviv, 1993). In muscle, insulin/ IGF-1 stimulates the Na+-K+ pump (Dørup and Clausen, 1995). The accumulation of Mg2+ caused by insulin/IGF-1 can occur even if the Mg2+-requiring (Na+-K+) ATPase is impaired by ouabain. Mg2+ efflux depends on the extent of the Na+ gradient across the membrane. Wasserman et al. reported that Na+/Mg2+ exchange can also occur in the reverse direction (Wasserman et al., 1986). Yoshimura et al. reported that Na+/Mg2+ exchange might be involved in the regulation of [Mg2+]i in human platelets (Yoshimura et al., 1995). Amiloride, an inhibitor of Na+-H+ antiport, did not block the increasing effect of insulin/IGF-1 on [Mg2+]i. These data suggest that Mg2+ influx with insulin/IGF-1 from extracellular
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+
2+
space is independent of Na -H antiport. The influx pathway for [Mg ]i by insulin/IGF1 is uncertain. Insulin/IGF-1 translocate Mg2+ from the extracellular space to intracellular space and these effects are affected by external sodium and potassium. ACKNOWLEDGMENTS This study was supported by Research Grants from the Morinaga Hoshikai and the Mami Mizutani Foundations.
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