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THU0110 V-atpase inhibitor bafilomycin a1 reduces proton secretion in fibroblasts
  1. B Stein1,
  2. T Pap2,
  3. M George1,
  4. WJ Parak1,
  5. O Müller3,
  6. S Gay2,
  7. WK Aicher3
  1. 1Institute for Applied Physics, LMU München, Munich
  2. 2WHO Rheumatology Res. Centre, University Hospital, Zurich, Switzerland
  3. 3Centre Orthopedic Surgery, University Medical School, Tübingen, Germany

Abstract

Background Metabolic activation of synovial fibroblasts (SF,1), prosthesis loosening fibroblasts (PLF,2) and osteoblasts (OB,3) results in an enhanced acid secretion as determined by cytosensor microphysiometer. In addition, PLF were shown to degrade bone in absence of infiltrating macrophages or osteoclasts.

Objectives We were interested to study if the proton secretion was associated with the activity of specific proton pump, especially with the activity of V-type H ± ATPases.

Methods Fibroblasts or osteoblasts were expanded in complete medium containing 10% FCS and antibiotics. For cytosensor analysis cells were seeded at 30 000 cells/ml in microcups, mounted into the cytosensor. For metabolic activation the medium was enriched with cytokines such as TNF-alpha, IL-1 (Roche) or different agents such as ionomycin or PMA (Calbiochem) at different concentrations. To block V-ATPase activity, cells were incubated with Bafilomycin A1 or Amiloride (Calbiochem).

Results Metabolic activation as determined by enhanced acid secretion was observed upon stimulation of the fibroblasts with TNF-alpha, IL-1, PDGF, ionomycin or PMA but not with IL-6 or bFGF. Osteoblasts were activated by TNF-alpha, ionomycin and PMA; bFGF, IL-1, IL-6 or PDGF are not tested yet. Acid secretion occurred as early as 20–30 min. after initial stimulation. Under identical conditions, immortalised synovial fibroblast clones secreted less acid when compared to normal fibroblasts. As metabolic activation stimulates glycolysis, the acidification may be simply a consequence of carbohydrate catabolism generating acetate, lactate or carbonate. Therefore fibroblasts were incubated in medium containing Bafilomycin A1, a specific V-type H ± ATPase blocker. Bafilomycin reduced the proton secretion in a time course experiment within 20 min. irreversibly. Interestingly, Amiloride inhibited the proton secretion reversibly.

Conclusion Metabolic activation of fibroblasts or osteoblasts by pro-inflammatory cytokines results in enhanced acid secretion. As pro-inflammatory cytokines enhance the expression of matrix degrading proteases as well, we conclude that the acidification of the pericellular milieu by mesenchymal cells accelerates matrix degradation. In addition, we provide for the first time experimental evidence that the acid secreted upon metabolic activation is not only a product of anaerobe (lactate, acetate) or aerobe (carbonate) glycolysis, but may be associated with a specific V-ATPase located in vacuolar membranes in the cytoplasm or on the surface of the cells. Preliminary data corroborate this conclusion, since a V-ATPase was located on the cell surface by immunohistochemistry and a V-ATPase encoding mRNA was detected by RT/PCR in the respective cells.

References

  1. Parak, et al. Metabolic activation stimulates acid production in synovial fibroblasts. J Rheumatol. 2000;27:2312

  2. Sainsbury, et al. Cathepsin K expression by activated fibroblasts at the bone interface of the pseudosynovium in aseptic prosthesis loosening. Proc. ORS Conv. Anaheim # 27, 1999

  3. Mast A. Charakterisierung von Knochenzellen mit biophysikalischen Methoden auf zellulärem Niveau, Master Thesis, Fakult. of Physics, University of Tübingen, 1999

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