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The effect of IL-1 on acid extrusion from bovine articular chondrocytes

INTRODUCTION Acidity has been shown to be a determinant of matrix turnover [1], and has been implicated in cartilage degeneration [2]. Our previous work has shown that articular chondrocytes regulate intracellular pH (pHi) using the amiloride sensitive Nax H exchanger [3]. There is also a small invariant component inhibited by NBD-Cl, which represents a H-ATPase. The specific cytokine interleukin-1 (IL-1) is found at enhanced levels in synovial fluid, and the extracellular matrix of arthritic tissue. IL-1 inhibits proteoglycan synthesis and enhances matrix metalloproteinase (MMP) activity. Induction of nitric oxide (NO) has been implicated in some of the actions of IL-1 [5]. The present study characterises how IL-1 can modify pHi regulation in chondrocytes and the involvement of NO pathways in these events. METHODS Chondrocytes were isolated from bovine metacarpophalangeal cartilage using collagenase (1mg ml, 37C, 18h) in DMEM in the absence or presence of IL1α (0.5ng ml), IL-1α and L-NAME (N(G)-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor; 1mM), or SNAP (S-nitroso-N-acetylpenicillamine, nitric oxide donor; 200μM, 6h). pHi was measured in cells loaded with the fluorescent indicator BCECF (BCECF-AM, 10μM, 37C, 30min) using a spectrofluorimeter (EX = 490nm/439nm, EM = 535nm). The ratio 490nm/439nm was calibrated using nigericin (3μM) in a high-K solution [3]. The cells were acidified using ammonium prepulse (20mM NH4Cl, 15min) and proton equivalent fluxes (JH; mM min ; [6]) were calculated for the first 180s of recovery in HEPES buffered solutions (pH 7.4). The effect of treatment with amiloride (1mM, NHE non-specific), HOE-694 (60μM, NHE-1 specific action), NBD-Cl (100μM), or removal of extracellular Na ions was characterised. 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 [7]. RESULTS Recovery from intracellular acidification was strongly dependent on extracellular Na ions, and was significantly inhibited in cells treated with amiloride (Table 1). The small residual acid efflux, recorded on removal of extracellular Na ions, was inhibited by treatment with NBD-Cl. Overnight exposure to IL-1 significantly enhanced acid extrusion. The IL-1 enhancement was significantly attenuated in cells exposed to NBD-Cl, while a smaller but nonetheless significant reduction was also observed in Na-free solutions or following treatment with amiloride (Table 1).