Homeostasis of pH in equine chondrocytes

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Online Journal of Veterinary Research©

Volume 7:59-70, 2003. Redacted 2018.

Fairfax TPA^a, Davies ME^b, Milner P^b, Gibson JS^c, Wilkins RJ^a

^aUniversity of Oxford, University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK; ^bUniversity of Cambridge, Equine Orthopaedic Research Group & ^cCentre for Veterinary Science, Department of Clinical Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, UK.Author to whom correspondence should be addressed: J. S. Gibson Telephone: 00 44 (0) 1223 337638 Fax: 00 44 (0) 1223 337610, Email: jsg1001@cam.ac.uk

ABSTRACT

Fairfax TPA, Davies ME, Milner P, Gibson JS, Wilkins RJ. Homeostasis of pH in equine chondrocytes, Onl J Vet Res., 7:59-70, 2003. pH homestasis in articular chondrocytes affects synthesis and degradation of the cartilage matrix and Ca²⁺ balance. However, the mechanisms by which equine chondrocytes regulate pH remain unknown. Intracellular pH (pH_i) was determined with the fluorophore BCECF and NH₄Cl was used to alter pH_i. pH recovery was monitored in the presence and absence of inhibitors of Na⁺/H⁺ exchange (NHE) and H⁺-ATPase, and in HEPES- and HCO₃^-/CO₂-buffered saline (HBS and BBS, respectively). In some experiments, [Ca²]_i was measured using Fura-2. Under steady state conditions in HBS, pH_i in equine chondrocytes was 7.18 + 0.02 S.E.M., n = 6). On acidification, pH recovered with a time constant (t) of 26 + 8 s (n = 3). Recovery was inhibited by removal of extracellular Na⁺ or application of HOE694, a specific inhibitor of the NHE-1 isoform. Cells incubated in BBS showed more rapid pH recovery, partially (40%) inhibited by SITS. For acidified cells suspended in BBS, a small but consistent recovery of pH_i occurred in the presence of HOE694. Intrinsic buffering capacity was approximately 20 mmol.(l cells)^-1 pH unit^-1 at steady-state pH_i and was increased at more acidic pH_i. Finally, when pH was increased (to about 7.7) with NH₄Cl, there was a parallel increase in [Ca²⁺]_i -(of about 60 nM). The marked sensitivity to HOE694 of recovery from acidosis in both HBS and BBS is consistent with a major role for NHE-1 in pH_i homeostasis in equine chondrocytes. There are also small but significant contributions from H⁺-ATPase and HCO₃^- dependent processes, probably including both HCO₃^-/Cl^- exchange and Na⁺-dependent HCO₃^- transport. Ca²⁺ homeostasis is affected by pH_i. A more complete understanding of pH homeostasis in equine chondrocytes may allow the development of future therapeutic regimes to protect against joint disease.

KEY WORDS: chondrocytes – pH – Na⁺/H⁺ exchange.

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