Regulation of Intracellular pH by Bovine Intervertebral Disc Cells

Abstract

Extracellular acidity is an important determinant of intervertebral disc matrix turnover, possibly exerting effects through changes of intracellular pH_i (pH). There is, however, little information concerning the ways in which these cells regulate their pH_i. Fluorimetric techniques have been used in the present study to measure pH_i in isolated intervertebral disc cells, and to characterise the membrane transport pathways by which it is regulated. Nucleus pulposus cells were obtained from bovine intervertebral discs by standard enzymatic digestion techniques, and loaded with the pH-sensitive fluoroprobe BCECF. Resting pH_i was approximately 6.7 for cells suspended in either HEPES-buffered (HBS) or CO₂/HCO₃^–-buffered (BBS) media. Intrinsic buffering capacity was approximately 19 mM pH unit^–1 in HBS and was increased when cells were suspended in BBS. A combination of ion substitution and inhibitor studies for cells at steady-state pH_i or acidified by exposure to NH₄Cl revealed that in HBS Na⁺ × H⁺ exchange and an H⁺-ATPase extrude acid from these cells. Only one of these two systems, the Na⁺ × H⁺ exchanger, exhibited a sensitivity to pH_i, identifying it as the regulator of pH_i under these conditions. In BBS, an additional pathway which was dependent on extracellular Na⁺, extracellular HCO₃^– and intracellular Cl^– was detected. These properties are consistent with the four ion HCO₃^–-dependent transporter, although the cation-rich, anion-poor extracellular matrix of the intervertebral disc means that such a pathway has only a marginal role in disc cell pH_i regulation.