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Bradykinin Increases Intracellular Free Ca2+Concentration and Promotes Insulin Secretion in the Clonal β-Cell Line, HIT-T15
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 577–580 (1996)
0638
Bradykinin Increases Intracellular Free Ca2+ Concentration and Promotes Insulin Secretion in the Clonal b-Cell Line, HIT-T15 Yutaka Saito, Masakatsu Kato, Yuzuru Kubohara, Isao Kobayashi, and Kazuhiko Tatemoto1 Department of Laboratory Medicine and Clinical Laboratory Center, School of Medicine and Department of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan Received March 18, 1996 We have examined the effects of bradykinin (BK) on both the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in the hamster b-cell line, HIT-T15 cells. BK evoked a rise in [Ca2+]i in a dose-dependent manner. This response was suppressed by neomycin, suggesting that BK mobilizes Ca2+ from intracellular store via promotion of the phosphatidyl inositol turnover. Furthermore, BK also evoked insulin secretion. Both the BK-evoked rise in [Ca2+]i and insulin secretion were suppressed by the BK2 receptor antagonist, but not by the BK1 receptor antagonist. These results indicate that BK increases [Ca2+]i via BK2 receptor, thereby promoting insulin secretion in HIT-T15 cells. © 1996 Academic Press, Inc.
Bradykinin (BK) is a bioactive peptide that mediates the activation of sensory and sympathetic neurons, enhances smooth muscle contraction, increases vascular tone, stimulates glandular secretion, and modulates pain associated with cardiac anoxia, inflammation, and rheumatoid diseases (1–3). In many cell types such as neurons (4), mesangial cells (5), and aortic endothelial cells (6), BK induces an increase in the intracellular free calcium concentration ([Ca2+]i) at least partly via promotion of phosphatidyl inositol (PI) turn-over. Recently, the involvement of BK in the regulation of pancreatic functions has been suggested. Northern blot analysis first revealed the presence of mRNA for BK2 (B2) receptors in rat pancreas (7), and a high level of B2 receptor was found also in human pancreas by a competitive PCR method (8). Quite recently, Yang et al. reported that BK evoked insulin release from the perfused rat pancreas (9). It is therefore likely that BK plays some role(s) in the regulation of insulin secretion in the pancreas. However, it is not clear if BK directly acts on pancreatic b-cells nor how this peptide regulates insulin secretion in the pancreas. In this study, in order to assess the above questions, we examined the effects of BK on the insulin producing cell line, HIT-T15 cells, and show here that BK increases [Ca2+]i and promotes insulin secretion in this cell line. MATERIALS AND METHODS Cell line and chemicals. Hamster insulinoma cell line HIT-T15 (HIT) (10,11) was used in this study. Bradykinin (BK), des-Arg9-[Leu8]-bradykinin (B1 antagonist) and D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin (B2 antagonist) were purchased from Peptide Institute Inc. (Osaka, Japan). Neomycin and fura-2/AM were from Wako Pure Chemical Industries (Osaka, Japan). Preparation of HIT cells. HIT cells were cultured in the RPMI1640 medium (GIBCO, N.Y.) supplemented with 20 mM HEPES (pH 7.4), 5 mM NaHCO3, 25 mg/l penicillin, 50 mg/l streptomycin, and 10% fetal calf serum at 37 C under 5% CO2-95% air. For calcium-imaging experiments, the cells were collected after trypsin treatment, plated on poly-D-lysine coated (0.2 mg/ml) coverslips, and cultured for 2 days in the RPMI1640 medium. Measurements of [Ca2+]i in single HIT cells. HIT cells plated on coverslips were loaded with fura-2/AM (1 mg/ml) for 30 min in a perifusion medium and then placed on a flow-through chamber mounted on the stage of a microscope. The perifusion medium contained (mM) NaCl 135, KCl 5.0, CaCl2 2.5, MgCl2 0.8, HEPES 20, glucose 5.5, BSA 0.1% (pH was 1
Corresponding author. Department of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. Fax: 81-272-20-8849. Abbreviations: BK, bradykinin; [Ca2+]i, intracellular free calcium concentration; PI, phosphatidyl inositol. 577 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
adjusted to 7.35 with NaOH). Argus 100 system (Hamamatsu Photonics, Japan) was used for all dynamic video imaging and image processing. Excitation wavelengths of 340 nm and 380 nm were altered by means of a computer-controlled rotating filter wheel between the mercury ultraviolet source and the microscope. Emission light at 510 nm was passed to an image intensifying charge coupled device camera (C2400-87, Hamamatsu Photonics, Japan). The resulting image was averaged (8 images), digitized, captured, and stored. The time resolution between ratio frames was 4 sec. Ratios were converted to Ca2+ concentrations according to Grynkiewicz et al (12). Measurements of insulin release. HIT cells were plated in 12-well plates (106 cells/well) and were cultured for 2 days in the RPMI1640 medium. The cells were then washed twice with the perifusion medium. After this, they were incubated for 20 min at 37 C in two solutions of the same medium containing 100 nM BK with and without BK antagonist (1 mM). At the end of incubation period, the medium was collected from each well and the insulin contents in the media were determined by using an insulin assay kit (Pharmacia, Uppsala, Sweden). Statistical analysis. For quantitative analysis, the mean [Ca2+]i was calculated for the regions of interest. Data are expressed as means ± S.E.M. Statistical analysis was performed with Student’s t-test following the analysis of variance.
RESULTS Effects of BK on [Ca ]i in HIT cells. BK at a concentration range of between 10 pM and 1 mM evoked a rise in [Ca2+]iin HIT cells in a dose-dependent manner (Fig. 1). The proportion of the cells responsive to BK was about 80 % at a concentration of BK $ 1 nM. BK (10 nM)-induced rise in [Ca2+]i was not significantly affected by the removal of the extracellular Ca2+ (data not shown), but was suppressed by the treatment of the cells with neomycin (Fig. 2), an inhibitor of phosphatidyl inositol (PI) turn-over (13). Neomycin at 2 mM and 10 mM suppressed the BK-evoked peak values in [Ca2+i by 46 ± 7% and 77 ± 4%, respectively. Although high concentrations (e.g. 10 mM) of neomycin may have non-specific effects, these results suggest that BK promotes Ca2+ mobilization from intracellular Ca2+ store(s) at least partly via PI turn-over. Effects of BK receptor antagonists on the BK-induced rise in [Ca2+]i. The effects of B1 and B2 antagonists were examined on 10 mM BK-evoked rise in [Ca2+]i in HIT cells. B2 antagonist dose-dependently suppressed the response at a concentration range between 1 nM and 1 mM (Fig. 3). B2 antagonist at 1 mM suppressed the peak value of the response by 91 ± 2%, whereas B1 antagonist (1mM) did not suppress the response. Effects of BK on insulin secretion in HIT cells. We also examined whether BK affected insulin secretion in HIT cells. BK (100 nM) induced an approximately 2-fold increase in insulin secretion in the presence of 5.5 mM glucose during a 20-min incubation period (Fig. 4). It should be noted here that changing the glucose concentration to 2.8 mM or to 8 mM did not affect the action of BK 2+
FIG. 1. Effect of BK on [Ca2+]i in single HIT cells. The fura-2-loaded cells were stimulated with various concentrations of BK. (A) Representative traces are shown. (B) Data are given as means ± S.E.M. The numbers of the cells tested are shown in the figure. Application of BK is indicated by a horizontal bar above each trace. 578
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Effect of neomycin on BK-induced rise in [Ca2+]i in HIT cells. The fura-2-loaded cells were treated with various concentrations of neomycin (NM) for 30 min prior to the addition of 10 nM BK. (A) Representative traces are shown. (B) Data are given as the means ± S.E.M. The number of the cells tested is shown in the figure. Application of BK and NM is indicated by horizontal bars above each trace. **, P < 0.01 vs. control.
on insulin secretion (data not shown). B2 antagonist (1 mM) suppressed this insulin secretion by 35.4 ± 8.1%, whereas B1 antagonist (1 mM) had no effect on it. DISCUSSION In this study, we have shown for the first time that BK induces an increase in [Ca2+]i (Fig. 1) and promotes insulin secretion (Fig. 4) in the insulinoma HIT cells. Since the BK-induced calcium increase was not significantly altered by removal of extracellular Ca2+ (data not shown), and was reduced by the treatment with neomycin (Fig. 2), BK would promote Ca2+ mobilization from some intracellular store(s) at least partly via PI turn-over. The study with the antagonists to B1 and B2
FIG. 3. Effects of BK receptor antagonists on the BK-induced rise in [Ca2+]i in HIT cells. The fura-2-loaded cells were incubated with various concentrations of B1 or B2 antagonist prior to the stimulation with 10 nM BK. (A) Representative traces are shown. (B) Data are given as the means ± S.E.M. (v) with B2 antagonist, (V) with B1 antagonist. The number of the cells tested is shown in the figure. Application of BK and BK antagonist is indicated by horizontal bars above each trace. **, P < 0.01 vs. control (without antagonist). 579
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FIG. 4. Effect of BK on insulin secretion in HIT cells. The cells were incubated without (control) or with 100 nM BK, or with 100 nM BK in the presence of 1 mM B1 antagonist (B1) or B2 antagonist (B2). Each bar represents the mean ± S.E.M. of triplicate determinations of two independent experiments. Two other experiments gave similar results. **, P < 0.01. *, P < 0.05.
receptors (Fig. 3,4) indicated that both the increase in [Ca2+]i and the promotion of insulin secretion by BK are exerted via B2 receptors in this cell line, which agrees well with the fact that B2 receptors mediate most physiological effects of BK (3,7). Although the molecular mechanism of insulin secretion involving [Ca2+]i is not clear at present, it is likely that BK promotes insulin secretion via an increase in [Ca2+]i in HIT cells. Recently, Yang et al. reported that BK (0.01–1 mM) evoked insulin release from the perfused rat pancreas and this response was abolished by a specific B2 receptor antagonist, HOE-140 (9). In their system, however, cellular interactions within the islets or neural interactions cannot be neglected. In this study, using HIT cells as a model for pancreatic b-cells, we have suggested that BK may act directly on b-cells and promotes insulin secretion in vivo. At any rate, further experimentation is needed for the elucidation of the cellular and molecular mechanisms of BK action in pancreatic b-cells, and HIT cells should provide a useful model system suitable for the purpose. ACKNOWLEDGMENTS We are very grateful to Dr. K. Ishikawa for useful discussion and critical reading of the manuscript. This work was supported in part by a Grant-in-Aid 04404084 for Scientific Research from the ministry of Education, Science and Culture of Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Dray, A., and Perkins, M. (1993) TINS 16, 99–103. Miller, R. J. (1987) TINS 10, 226–228. Hall, J. M. (1992) Pharmacol. Ther. 56, 131–190. Thayer, S. A., Perney, T. M., and Miller, R. J. (1988) J. Neurosci. 8, 4089–4097. Bascands, J. L., Pecher, C., Bompart, G., Rakotoarivony, J., Tack, J. L., and Girolami, J. P. (1994) Am. J. Physiol. 267, F871–878. Himmel, H. M., Rasmusson, R. L., and Strauss, H. C. (1994) Am. J. Physiol. 267, C1338–1350. Mceachern, A. E., Shelton, E. R., Bhakta, S., Obernolte, R., Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R. W., and Jarnagain, K. (1991) Proc. Natl. Acad. Sci. USA 88, 7724–7728. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D., and Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, 260–268. Yang, C., and Hsu, W. H. (1995) Am. J. Physiol. 268, E1027–1030. Santerre, R. F., Cook, R. A., Crisel, R. M. D., Sharp, J. D., Schmidt, R. J., Williams, D. C., and Wilson, C. P. (1981) Proc. Natl. Acad. Sci. USA 78, 4339–4343. Hill, R. S., and Boyd, A. E. (1985) Diabetes 34, 115–120. Grynkiewicz, G., Peonie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. Carney, D. H., Scott, D. L., Gordon, E. A., and Labelle, E. F. (1985) Cell 42, 479–488.
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221, 577–580 (1996)
0638
Bradykinin Increases Intracellular Free Ca2+ Concentration and Promotes Insulin Secretion in the Clonal b-Cell Line, HIT-T15 Yutaka Saito, Masakatsu Kato, Yuzuru Kubohara, Isao Kobayashi, and Kazuhiko Tatemoto1 Department of Laboratory Medicine and Clinical Laboratory Center, School of Medicine and Department of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan Received March 18, 1996 We have examined the effects of bradykinin (BK) on both the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in the hamster b-cell line, HIT-T15 cells. BK evoked a rise in [Ca2+]i in a dose-dependent manner. This response was suppressed by neomycin, suggesting that BK mobilizes Ca2+ from intracellular store via promotion of the phosphatidyl inositol turnover. Furthermore, BK also evoked insulin secretion. Both the BK-evoked rise in [Ca2+]i and insulin secretion were suppressed by the BK2 receptor antagonist, but not by the BK1 receptor antagonist. These results indicate that BK increases [Ca2+]i via BK2 receptor, thereby promoting insulin secretion in HIT-T15 cells. © 1996 Academic Press, Inc.
Bradykinin (BK) is a bioactive peptide that mediates the activation of sensory and sympathetic neurons, enhances smooth muscle contraction, increases vascular tone, stimulates glandular secretion, and modulates pain associated with cardiac anoxia, inflammation, and rheumatoid diseases (1–3). In many cell types such as neurons (4), mesangial cells (5), and aortic endothelial cells (6), BK induces an increase in the intracellular free calcium concentration ([Ca2+]i) at least partly via promotion of phosphatidyl inositol (PI) turn-over. Recently, the involvement of BK in the regulation of pancreatic functions has been suggested. Northern blot analysis first revealed the presence of mRNA for BK2 (B2) receptors in rat pancreas (7), and a high level of B2 receptor was found also in human pancreas by a competitive PCR method (8). Quite recently, Yang et al. reported that BK evoked insulin release from the perfused rat pancreas (9). It is therefore likely that BK plays some role(s) in the regulation of insulin secretion in the pancreas. However, it is not clear if BK directly acts on pancreatic b-cells nor how this peptide regulates insulin secretion in the pancreas. In this study, in order to assess the above questions, we examined the effects of BK on the insulin producing cell line, HIT-T15 cells, and show here that BK increases [Ca2+]i and promotes insulin secretion in this cell line. MATERIALS AND METHODS Cell line and chemicals. Hamster insulinoma cell line HIT-T15 (HIT) (10,11) was used in this study. Bradykinin (BK), des-Arg9-[Leu8]-bradykinin (B1 antagonist) and D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin (B2 antagonist) were purchased from Peptide Institute Inc. (Osaka, Japan). Neomycin and fura-2/AM were from Wako Pure Chemical Industries (Osaka, Japan). Preparation of HIT cells. HIT cells were cultured in the RPMI1640 medium (GIBCO, N.Y.) supplemented with 20 mM HEPES (pH 7.4), 5 mM NaHCO3, 25 mg/l penicillin, 50 mg/l streptomycin, and 10% fetal calf serum at 37 C under 5% CO2-95% air. For calcium-imaging experiments, the cells were collected after trypsin treatment, plated on poly-D-lysine coated (0.2 mg/ml) coverslips, and cultured for 2 days in the RPMI1640 medium. Measurements of [Ca2+]i in single HIT cells. HIT cells plated on coverslips were loaded with fura-2/AM (1 mg/ml) for 30 min in a perifusion medium and then placed on a flow-through chamber mounted on the stage of a microscope. The perifusion medium contained (mM) NaCl 135, KCl 5.0, CaCl2 2.5, MgCl2 0.8, HEPES 20, glucose 5.5, BSA 0.1% (pH was 1
Corresponding author. Department of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. Fax: 81-272-20-8849. Abbreviations: BK, bradykinin; [Ca2+]i, intracellular free calcium concentration; PI, phosphatidyl inositol. 577 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
adjusted to 7.35 with NaOH). Argus 100 system (Hamamatsu Photonics, Japan) was used for all dynamic video imaging and image processing. Excitation wavelengths of 340 nm and 380 nm were altered by means of a computer-controlled rotating filter wheel between the mercury ultraviolet source and the microscope. Emission light at 510 nm was passed to an image intensifying charge coupled device camera (C2400-87, Hamamatsu Photonics, Japan). The resulting image was averaged (8 images), digitized, captured, and stored. The time resolution between ratio frames was 4 sec. Ratios were converted to Ca2+ concentrations according to Grynkiewicz et al (12). Measurements of insulin release. HIT cells were plated in 12-well plates (106 cells/well) and were cultured for 2 days in the RPMI1640 medium. The cells were then washed twice with the perifusion medium. After this, they were incubated for 20 min at 37 C in two solutions of the same medium containing 100 nM BK with and without BK antagonist (1 mM). At the end of incubation period, the medium was collected from each well and the insulin contents in the media were determined by using an insulin assay kit (Pharmacia, Uppsala, Sweden). Statistical analysis. For quantitative analysis, the mean [Ca2+]i was calculated for the regions of interest. Data are expressed as means ± S.E.M. Statistical analysis was performed with Student’s t-test following the analysis of variance.
RESULTS Effects of BK on [Ca ]i in HIT cells. BK at a concentration range of between 10 pM and 1 mM evoked a rise in [Ca2+]iin HIT cells in a dose-dependent manner (Fig. 1). The proportion of the cells responsive to BK was about 80 % at a concentration of BK $ 1 nM. BK (10 nM)-induced rise in [Ca2+]i was not significantly affected by the removal of the extracellular Ca2+ (data not shown), but was suppressed by the treatment of the cells with neomycin (Fig. 2), an inhibitor of phosphatidyl inositol (PI) turn-over (13). Neomycin at 2 mM and 10 mM suppressed the BK-evoked peak values in [Ca2+i by 46 ± 7% and 77 ± 4%, respectively. Although high concentrations (e.g. 10 mM) of neomycin may have non-specific effects, these results suggest that BK promotes Ca2+ mobilization from intracellular Ca2+ store(s) at least partly via PI turn-over. Effects of BK receptor antagonists on the BK-induced rise in [Ca2+]i. The effects of B1 and B2 antagonists were examined on 10 mM BK-evoked rise in [Ca2+]i in HIT cells. B2 antagonist dose-dependently suppressed the response at a concentration range between 1 nM and 1 mM (Fig. 3). B2 antagonist at 1 mM suppressed the peak value of the response by 91 ± 2%, whereas B1 antagonist (1mM) did not suppress the response. Effects of BK on insulin secretion in HIT cells. We also examined whether BK affected insulin secretion in HIT cells. BK (100 nM) induced an approximately 2-fold increase in insulin secretion in the presence of 5.5 mM glucose during a 20-min incubation period (Fig. 4). It should be noted here that changing the glucose concentration to 2.8 mM or to 8 mM did not affect the action of BK 2+
FIG. 1. Effect of BK on [Ca2+]i in single HIT cells. The fura-2-loaded cells were stimulated with various concentrations of BK. (A) Representative traces are shown. (B) Data are given as means ± S.E.M. The numbers of the cells tested are shown in the figure. Application of BK is indicated by a horizontal bar above each trace. 578
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FIG. 2. Effect of neomycin on BK-induced rise in [Ca2+]i in HIT cells. The fura-2-loaded cells were treated with various concentrations of neomycin (NM) for 30 min prior to the addition of 10 nM BK. (A) Representative traces are shown. (B) Data are given as the means ± S.E.M. The number of the cells tested is shown in the figure. Application of BK and NM is indicated by horizontal bars above each trace. **, P < 0.01 vs. control.
on insulin secretion (data not shown). B2 antagonist (1 mM) suppressed this insulin secretion by 35.4 ± 8.1%, whereas B1 antagonist (1 mM) had no effect on it. DISCUSSION In this study, we have shown for the first time that BK induces an increase in [Ca2+]i (Fig. 1) and promotes insulin secretion (Fig. 4) in the insulinoma HIT cells. Since the BK-induced calcium increase was not significantly altered by removal of extracellular Ca2+ (data not shown), and was reduced by the treatment with neomycin (Fig. 2), BK would promote Ca2+ mobilization from some intracellular store(s) at least partly via PI turn-over. The study with the antagonists to B1 and B2
FIG. 3. Effects of BK receptor antagonists on the BK-induced rise in [Ca2+]i in HIT cells. The fura-2-loaded cells were incubated with various concentrations of B1 or B2 antagonist prior to the stimulation with 10 nM BK. (A) Representative traces are shown. (B) Data are given as the means ± S.E.M. (v) with B2 antagonist, (V) with B1 antagonist. The number of the cells tested is shown in the figure. Application of BK and BK antagonist is indicated by horizontal bars above each trace. **, P < 0.01 vs. control (without antagonist). 579
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Effect of BK on insulin secretion in HIT cells. The cells were incubated without (control) or with 100 nM BK, or with 100 nM BK in the presence of 1 mM B1 antagonist (B1) or B2 antagonist (B2). Each bar represents the mean ± S.E.M. of triplicate determinations of two independent experiments. Two other experiments gave similar results. **, P < 0.01. *, P < 0.05.
receptors (Fig. 3,4) indicated that both the increase in [Ca2+]i and the promotion of insulin secretion by BK are exerted via B2 receptors in this cell line, which agrees well with the fact that B2 receptors mediate most physiological effects of BK (3,7). Although the molecular mechanism of insulin secretion involving [Ca2+]i is not clear at present, it is likely that BK promotes insulin secretion via an increase in [Ca2+]i in HIT cells. Recently, Yang et al. reported that BK (0.01–1 mM) evoked insulin release from the perfused rat pancreas and this response was abolished by a specific B2 receptor antagonist, HOE-140 (9). In their system, however, cellular interactions within the islets or neural interactions cannot be neglected. In this study, using HIT cells as a model for pancreatic b-cells, we have suggested that BK may act directly on b-cells and promotes insulin secretion in vivo. At any rate, further experimentation is needed for the elucidation of the cellular and molecular mechanisms of BK action in pancreatic b-cells, and HIT cells should provide a useful model system suitable for the purpose. ACKNOWLEDGMENTS We are very grateful to Dr. K. Ishikawa for useful discussion and critical reading of the manuscript. This work was supported in part by a Grant-in-Aid 04404084 for Scientific Research from the ministry of Education, Science and Culture of Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Dray, A., and Perkins, M. (1993) TINS 16, 99–103. Miller, R. J. (1987) TINS 10, 226–228. Hall, J. M. (1992) Pharmacol. Ther. 56, 131–190. Thayer, S. A., Perney, T. M., and Miller, R. J. (1988) J. Neurosci. 8, 4089–4097. Bascands, J. L., Pecher, C., Bompart, G., Rakotoarivony, J., Tack, J. L., and Girolami, J. P. (1994) Am. J. Physiol. 267, F871–878. Himmel, H. M., Rasmusson, R. L., and Strauss, H. C. (1994) Am. J. Physiol. 267, C1338–1350. Mceachern, A. E., Shelton, E. R., Bhakta, S., Obernolte, R., Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R. W., and Jarnagain, K. (1991) Proc. Natl. Acad. Sci. USA 88, 7724–7728. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D., and Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, 260–268. Yang, C., and Hsu, W. H. (1995) Am. J. Physiol. 268, E1027–1030. Santerre, R. F., Cook, R. A., Crisel, R. M. D., Sharp, J. D., Schmidt, R. J., Williams, D. C., and Wilson, C. P. (1981) Proc. Natl. Acad. Sci. USA 78, 4339–4343. Hill, R. S., and Boyd, A. E. (1985) Diabetes 34, 115–120. Grynkiewicz, G., Peonie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440–3450. Carney, D. H., Scott, D. L., Gordon, E. A., and Labelle, E. F. (1985) Cell 42, 479–488.
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