0925 - Characterisation of Lactic Acid Transport in Bovine Articular Chondrocytes

INTRODUCTION The avascular environment of the articular chondrocyte dictates that metabolism is largely by anaerobic pathways, with the consequent production of large quantities of lactic acid. The lactic acid produced by chondrocytes must diffuse from the cells, through the extracellular matrix to the synovial fluid and thereafter to capillaries and the systemic circulation. There are reports of high, graded concentrations of lactic acid within the cartilage matrix [1], and we have previously described inhibition of matrix turnover by extracellular lactic acid [2]. At physiological pH values, lactic acid exists predominantly as the ionic species H and lactate. These charged lipophobic species will not readily cross the plasma membrane of cells, and their exchange between the cytoplasm and extracellular solution is commonly mediated by a family of membrane-bound H-lactate cotransport carrier proteins, called the monocarboxylate transporters, or MCT. To date, seven MCT isoforms have been reported, with tissue-specific distribution. For highly glycolytic tissues, such as 'white' skeletal muscle fibres and leukocytes, MCT-4 is the predominant isoform. This protein mediates H-lactate efflux from the cell, and demonstrates a low affinity for lactate and a high transport capacity, properties appropriate for cells producing large quantities of the substrate [3]. In the present study, the lactic acid transport properties of articular chondrocytes have been characterised. The efflux of lactic acid along with fixed charged density and the operation of other H extrusion proteins will be an important determinant of matrix pH, and hence macromolecular turnover. METHODS Chondrocytes were isolated from bovine metacarpophalangeal cartilage using collagenase (0.8mg ml, 18h) in DMEM. Intracellular pH (pHi) was measured in cells loaded with the fluorescent indicator BCECF (BCECF-AM, 10μM, 37°C, 30min) using a spectrofluorimeter (EX = 490nm/439nm, EM = 535nm). The ratio 490nm/439nm was calibrated using nigericin (3μM) in a high-K solution [4]. Cells (1 × 10 ml) were suspended in a HEPES-buffered saline (pH 7.4) and pHi recorded at steady-state, or following the addition of lactic acid (1-20mM) to the extracellular solution. The effects of treatment with the MCT inhibitor α-cyanohydroxycinnamic acid (α-CHC, 1-10mM) on steady-state pHi and on the response to lactic acid addition were also investigated. In experiments using α-CHC, the fluorescent signal was corrected for the concentration-dependent influence of the inhibitor on the fluorescent properties of BCECF. pHi changes were converted to acid equivalent fluxes (JH, mM min , ± SEM, n ≥ 3) using the equation JH = ∆pHi/∆t × buffering power (βi), where βi = 43.3pHi + 337 [5]. RESULTS Chondrocyte steady-state pHi was 7.1 7.2, consistent with previous reports for this cell type [4]. Addition of lactic acid (1-20mM) produced a rapid intracellular acidification (Figure 1a) which increased in magnitude with increasing concentrations of lactic acid, and saturated at the highest doses tested (Figure 1b). These data were used to determine Vmax and Km values for the influx, which were 19.2 ± 2 mmol (l cells) min and 10.5 ± 2.05mM respectively. For cells at steady-state pHi, treatment with α-CHC (1-10mM) produced an acidification, which was dependent on the concentration of α-CHC used (Figure 2). Treatment with α-CHC reduced the magnitude of acidification induced by addition of 10mM lactic acid (Figure 1b), with increasing concentrations of the inhibitor producing increasingly large effects. DISCUSSION For a predominantly anaerobic cell type, there must be an effective efflux from the cell of the large quantities of lactic acid produced if pHi is to be maintained. Given that the pKa of lactic acid means that it exists almost entirely as H and lactate ions at physiological pH, a carrier-mediated transport of these lipophobic species must be responsible for the efflux of the acid [3]. In this study, the pHi changes arising upon addition of lactic acid or of the MCT inhibitor α-CHC have been characterised in articular chondrocytes using an H-sensitive dye. Hence, the influx of H and lactate have been used here to assess the properties of a membrane transport system which would be expected to operate as an efflux pathway in vivo. Addition of increasing concentrations of lactic acid elicited a saturable intracellular acidification, indicative of a carrier-mediated process. This effect was inhibited by the well-characterised inhibitor of MCT, α-CHC, confirming that H-lactate cotransport mediated by this transporter is responsible for the acidification observed. The kinetic parameters of the MCT-mediated acidification are those of a high-capacity system (high Vmax) with low affinity (high Km) for lactate . Such properties are consistent with the MCT4 isoform, previously described in other glycolytic tissues [3], and are well-suited to the large quantities of lactic acid which must efflux from the anaerobic chondrocyte. The significant acidification of steady-state pHi which was observed upon addition of α-CHC indicates that there is a high basal level of lactic acid extrusion from these cells under resting conditions and is consistent with the high rates of acid production which have been reported by other workers [1]. This study is a first step towards the characterisation of a membrane transport process contributing to matrix pH, the activity of which will therefore dictate in part cartilage turnover. Figure 1 (a)