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Calcitonin inhibits proton extrusion in resorbing rat osteoclasts via protein kinase A

  • Endocrinology
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Abstract

Although calcitonin is well known to be a potent inhibitor of bone resorption, it remains unknown how it regulates osteoclastic H+ transport. In this study, we examined the effects of calcitonin on H+ extrusion in cultured rat resorbing osteoclasts using an intracellular pH (pHi) indicator, BCECF [2′7′-bis-(2-carboxyethyl)- 5-carboxyfluorescein]. Resorbing osteoclasts were identified by their formation of resorbing pits on calcium phosphate-coated quartz coverslips. Both basal pHi and H+ extrusion activity were significantly higher compared to non-resorbing osteoclasts. Two types of H+-extruding systems were identified by pharmacological and immunocytochemical means: a bafilomycin-A1- sensitive and an amiloride-sensitive system [H+ extrusion mediated by a vacuolar type proton pump (V-ATPase) and by a Na+/H+ exchanger (NHE), respectively]. Calcitonin inhibited both H+ extrusion activities in a dose-dependent manner and this action was mimicked by protein kinase A (PKA) activators, but not by protein kinase C (PKC) activators. Pretreatment with PKA inhibitors completely suppressed calcitonin-induced inhibition, whereas neither PKC inhibitors nor calcium chelators suppressed it. These results indicate that calcitonin inhibits H+ extrusion generated by V-ATPase and NHE via PKA activation. These inhibitory mechanisms of H+ transport by calcitonin are important for the regulation of bone resorption.

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Fig. 1A–D.
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Fig. 3A–C.

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References

  1. Bastani B, Ross FP, Kopito RR, Gluck SL (1996) Immunocytochemical localization of vacuolar H+-ATPase and Cl–-HCO3 − anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: effect of calcium-deficient diet on polar expression of the H+-ATPase pump. Calcif Tissue Int. 58:332–336

    Google Scholar 

  2. Blair HC, Teitelbaum SL, Ghiselli R, Gluck S (1989) Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245:855–857

    CAS  PubMed  Google Scholar 

  3. Casavola VS, Reshkin J, Murer H, Helmle-Kolb C (1992) Polarized expression of Na+/H+ exchange activity in clonal LLC-PK1 cells (Clone4 and PKE20) cells: Hormonal regulation. Pflügers Arch 418:282–289

    Google Scholar 

  4. Chakraborty M, Chatterjee D, Gorelick FS, Baron R (1994) Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and Na, K-ATPase in the kidney cell line LLC-PK1 by calcitonin. Proc Natl Acad Sci USA 91:2115–2119

    CAS  PubMed  Google Scholar 

  5. Chambers TJ, Magnus CJ (1982) Calcitonin alters behavior of isolated osteoclasts. J Pathol 136:27–39

    CAS  PubMed  Google Scholar 

  6. Chambers TJ, Fuller K, Darby JA (1987) Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone. J Cell Physiol 132:90–96

    CAS  PubMed  Google Scholar 

  7. Galvin RJ, Bryan P, Venugopalan M, Smith DP, Thomas JE (1998) Calcitonin responsiveness and receptor expression in porcine and murine osteoclasts: a comparative study. Bone 23:233–240

    Article  CAS  PubMed  Google Scholar 

  8. Gupta A, Edwards JC, Hruska KA (1996) Cellular distribution and regulation of NHE-1 isoform of Na-H exchanger in the avian osteoclast. Bone 18:87–95

    Article  CAS  PubMed  Google Scholar 

  9. Hall TJ, Chambers TJ (1990) Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption. J Cell Physiol 142:420–424

    CAS  PubMed  Google Scholar 

  10. Hall TJ, Schaeublin M, Chambers TJ (1992) Na+/H+-antiporter activity is essential for the induction, but not the maintenance of osteoclastic bone resorption and cytoplasmic spreading. Biochem Biophys Res Commun 188:1097–1103

    CAS  PubMed  Google Scholar 

  11. Kajiya H, Okabe K, Okamoto F, Tsuzuki T, Soeda H (2000) Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts. J Cell Physiol 183:83–90

    Article  CAS  PubMed  Google Scholar 

  12. Laitala-Leinonen T, Väänänen HK (1993) Proton channel part of vacuolar H+-ATPase and carbonic anhydrase II expression is stimulated in resorbing osteoclasts. J Bone Miner Res 8:119–126

    PubMed  Google Scholar 

  13. Laitala-Leinonen T, Howell ML, Dean GE, Väänänen HK (1996) Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts. Mol Biol Cell 7:129–142

    CAS  PubMed  Google Scholar 

  14. Lees RL Sabharwal VK, Heersche JNM (2001) Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films. Bone 28:187–194

    Article  CAS  PubMed  Google Scholar 

  15. Lehenkari PP, Laitala-Leinonen T, Linna TJ, Väänänen HK (1997) The regulation of pHi in osteoclasts is dependent on the culture substrate and on the stage of the resorption cycle. Biochem Biophys Res Commun 235:838–844

    Article  CAS  PubMed  Google Scholar 

  16. Miyauchi A, Hruska K, Greenfield EM, Duncan R, Alvares J, Barattolo R, Colucci S, Zambonin-Zallone A, Teitelbaum SL, Teti A (1990) Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly bone resorption. J Cell Biol 111:2543–2552

    CAS  PubMed  Google Scholar 

  17. Moonga BS, Alam AS, Bevis PJ, Avaldi F, Soncini R, Huang CL, Zaidi M (1992) Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin. J Endocrinol 132:241–249

    CAS  PubMed  Google Scholar 

  18. Mrkic B, Forgo J, Murer H, Helmle-Kolb C (1992) Apical and basolateral Na+/H+ exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH). J Membr Biol 130:250–217

    Google Scholar 

  19. Nanda A, Gukovskaya A, Tseng J, Grinstein S (1992) Activation of vacuolar-type proton pumps by protein kinase C: Role in neutrophil pH regulation. J Biol Chem 267:22740–22746

    CAS  PubMed  Google Scholar 

  20. Nordström T, Grinstein S, Brisseau GF, Manolson MF, Rotstein OD (1994) Protein kinase C activation accelerates proton extrusion by vacuolar-type H+-ATPase in murine peritoneal macrophages. FEBS Lett 350:82–86

    Article  PubMed  Google Scholar 

  21. Nordström T, Shrode LD, Rotstein OD, Romanek R, Goto T, Heersche JNM, Manolson MF, Brisseau GF, Grinstein S (1997) Chronic extracellular acidosis induced plasmalemmal vacuolar type H+ ATPase activity in osteoclasts. J Biol Chem 272:6354–6360

    Article  PubMed  Google Scholar 

  22. Okabe K, Takada K, Okamoto F, Kajiya H, Soeda H (1998) Calcitonin regulates channel activities in rat osteoclasts via protein kinase A [Abstract]. Bone 23 [Suppl.]:S336

  23. Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296–434

    CAS  PubMed  Google Scholar 

  24. Santhanagopal A, Chidiac P, Horne WC, Baron R, Dixon SJ (2001) Calcitonin rapidly increases Na+/H+ exchanger and metabolic acid production: effects mediated selectively by the C1a CT receptor isoform. Endocrinology 142:4401–4413

    CAS  PubMed  Google Scholar 

  25. Su Y, Chakraborty M, Nathanson MH, Baron R (1992) Differential effects of the 3′, 5′-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin. Endocrinology 131:1497–1502

    CAS  PubMed  Google Scholar 

  26. Suzuki H, Nakamura I, Takahashi N, Ikuhara T, Matsuzaki K, Isogai Y, Hori M, Suda T (1996) Calcitonin-induced changes in the associated with protein kinase A in osteoclasts. Endocrinology 137:4685–4590

    CAS  PubMed  Google Scholar 

  27. Teti A, Colucci S, Grano M, Argentino L, Zambonin-Zallone A (1992) Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts. Am J Physiol 263:C130–C139

    CAS  PubMed  Google Scholar 

  28. Weinman EJ, Dubinsky WP, Dinh Q, Steplock D, Shenolikar S (1989) Effect of limited trypsin digestion on the renal Na+/H+ exchanger and its regulation by cAMP-dependent protein kinase. J Membr Biol 109:233–241

    CAS  PubMed  Google Scholar 

  29. Zhen MH, Wysocki S, Wood DJ, Papadimitriou JM (1994) Carbonic anhydrase II gene transcript in cultured osteoclasts from neonatal rats: effect of calcitonin. Cell Tissue Res 276:7–13

    PubMed  Google Scholar 

  30. Zimolo Z, Wesolowski G, Rodan GA (1995) Acid extrusion is induced by osteoclast attachment to bone: Inhibition by alendronate and calcitonin. J Clin Invest 96:2277–2283

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Dr. A. Carl for valuable comments and English editing. We also are grateful to Dr. Kenji Kitamura for valuable comments. We thank Dr. Yoshinori Moriyama (University of Okayama, Okayama, Japan) for the gift of rabbit anti-V-ATPase antibody. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (no. 10771024 and Frontier Research Grant).

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Authors and Affiliations

  1. Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Tamura 2–15–1, 814–0193, Sawara-ku, Fukuoka, Japan

    Hiroshi Kajiya, Fujio Okamoto, Hidefumi Fukushima & Koji Okabe

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  1. Hiroshi Kajiya
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  2. Fujio Okamoto
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  3. Hidefumi Fukushima
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  4. Koji Okabe
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Correspondence to Hiroshi Kajiya.

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Kajiya, H., Okamoto, F., Fukushima, H. et al. Calcitonin inhibits proton extrusion in resorbing rat osteoclasts via protein kinase A. Pflugers Arch - Eur J Physiol 445, 651–658 (2003). https://doi.org/10.1007/s00424-002-0989-4

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  • DOI: https://doi.org/10.1007/s00424-002-0989-4

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