IntroductionNucleus pulposus cells are solely responsible for the production and maintenance of the extracellular matrix of the nucleus throughout life [31]. The cells have no direct contact with each other and rely on cell-matrix interactions and signals from the environment to maintain the function of the tissue. The cells synthesize all the matrix components [10,37,38,50] and also produce degradative enzymes including metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases or TIMPs), involved in matrix turnover [7,45,53]. The fine balance between synthesis of components and the breakdown of tissue determines the composition of the matrix; tissue integrity is thus dependent on the activity of the nucleus pulposus cells [53]. In vivo [20,43] and in vitro [14] environmental factors such as mechanical stress [30,34] and nutrient levels have been found to affect matrix composition, presumably by affecting macromolecule biosyntheAbstract It has long been known that very acidic conditions can be found in degenerate discs. The effect of these acid conditions on matrix turnover are, however, unknown. This study aimed to examine the effect of acidity on production of matrix components and on agents which break down the matrix in order to gain insight into the effect of pathological values of pH on matrix turnover. Cells were isolated from the nucleus of bovine discs and from bovine articular cartilage, embedded in alginate beads and cultured at pH levels maintained within the ranges seen in normal and pathological discs: pH 7.4-pH 6.3 for 48 h. Rates of sulphated glycosaminoglycan (GAG) and protein synthesis were measured, as well as rates of production of some agents involved in matrix breakdown, i.e. total and activated matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The results showed that acid conditions had a profound effect on cell matrix turnover; at pH 6.4, total production of most species measured was inhibited by more than 50% compared to production at pH 7.2; production of sulphated GAGs and of TIMP-1 fell by >90%. However production of active metalloproteinases by disc cells was relatively insensitive to pH, with activity at pH 6.3 not statistically different from that at pH 7.2. These findings indicate that exposure to acid conditions appears particularly deleterious for the disc matrix, as it inhibits the disc cells from synthesising functionally important molecules such as the sulphated GAGs but does not prevent the production of agents able to degrade matrix components. The low values of pH seen in some degenerate discs are thus likely to be involved in breakdown of the disc matrix.

The effects of extracellular and intracellular pH on matrix synthesis by isolated bovine chondrocytes were studied using radioisotope incorporation (35SO4 and 3H proline) and fluorescence techniques. Matrix synthesis exhibited a bimodal relation with decreased extracellular pH; with slight reductions (7.4 > pH > 7.1), synthesis increased (by up to 50%), whereas in more acidic media (pH < 7.1), synthesis was inhibited by up to 75% of control levels. The pHi was largely unchanged with extracellular acidity over the range producing stimulation of matrix synthesis but fell when exposed to the more acidic media shown to have an inhibitory action on matrix synthesis. The inhibition of matrix synthesis by lactic acid addition was unaffected by the lactic acid transporter alpha-CHC, suggesting H+ transport by this pathway is small. Direct imposition of a sustained intracellular acidosis (pHi = 6.65) using ammonium prepulse with amiloride inhibited matrix synthesis by about 20%. These results show that matrix synthesis by chondrocytes was affected by extracellular pH, an action which could not be entirely explained by changes to pHi.

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4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K + and Cl _ channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase.5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and downregulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca 2+ entry and depended on tyrosine kinase and mitogen-activated/ERKactivating kinase (MEK) activity.6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca 2+ , all of which play a crucial role in the volumeregulatory mechanism of human cervical cancer cells.

Objective. To determine the effects of varying O 2 on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O 2 tensions.Methods. Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2 ,7 -bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope 22 Na ؉ was used to determine the kinetics of Na ؉ /H ؉ exchange (NHE) and the activity of the Na ؉ /K ؉ pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2 ,7 -dichlorofluorescein diacetate.Results. The pH homeostasis was unaffected when comparing tissue maintained at 20% O 2 (the level in water-saturated air at 37°C) with that at 5% O 2 (which approximates the normal level in healthy cartilage); however, an O 2 tension of <5% caused a fall in intracellular pH (pH i ) and slowed pH i recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na ؉ /K ؉ pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H 2 O 2 , Co 2؉ , or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. Conclusion.The ability of articular chondrocytes to carry out pH homeostasis is compromised when O 2 tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O 2 , such as in chronic inflammation.