The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.
Cartilage matrix turnover is sensitive to changes in intracellular pH (pHi) and previous studies have shown that articular chondrocytes regulate pHi predominantly using an amiloride-sensitive Na+ / H+ exchanger (NHE) with hallmark properties of the housekeeper isoform NHE1. Immunoblotting studies have, however, demonstrated that bovine chondrocytes express both the NHE1 and NHE3 isoforms of Na+ / H+ exchange. In the present study the effect of exposure to serum on acid extrusion from chondrocytes has been studied. The pH-sensitive fluoroprobe BCECF was used to measure pHi in isolated bovine articular chondrocytes, and proton equivalent membrane transporters were characterised for cells isolated in the absence and presence of 5% fetal bovine serum (FBS). The contribution of NHE isoforms to acid extrusion, following ammonium-induced acidification, was assessed using a combination of ion substitution and the specific NHE1 inhibitor HOE694. While Na+ -dependent acid extrusion was entirely inhibited by HOE694 in FBS untreated cells, the operation of both NHE1 and NHE3 was detected in cells exposed to FBS. In parallel, RT-PCR and immunohistochemistry experiments demonstrated both NHE1 and NHE3 mRNA and expression of both proteins respectively. While serum growth factors are virtually excluded from healthy cartilage they could permeate a damaged matrix. The regulatory characteristics of NHE3 are distinct from NHE1 so that an altered pattern of response to components of mechanical stress such as hyperosmolarity may be associated with increased access of growth factors in diseased tissue.
Incubation with serum modulates the transporters that regulate intracellular pH (pH(i)) in articular chondrocytes, upregulating acid extrusion by Na(+)-H(+) exchange (NHE). There is stimulation of NHE1, together with induction of NHE3 activity. These isoforms exhibit differential responses to components of mechanical load experienced by chondrocytes during joint loading. The identity of the component(s) of serum responsible is unknown. A possibility, however, is insulin-like growth factor-1 (IGF-1), present in normal cartilage and found at enhanced levels in osteoarthritic tissue. In the present study, the effects of IGF-1 on pH(i) regulation have been characterized using fluorescence measurements of bovine articular chondrocytes, and the sensitivity of pH(i) regulation to hyperosmotic shock and raised hydrostatic pressure determined. For cells isolated in the absence of IGF-1, pH(i) recovery following acidification was predominantly mediated by NHE1. Recovery was enhanced when cells were incubated for 18 h with 20 ng mL(-1) IGF; this effect represented increased acid extrusion by NHE1, supplemented by NHE3 activity. NHE3 activity was not detected in IGF-1-treated cells that had been incubated with the protein synthesis inhibitor cycloheximide, although NHE1 activity was unaffected. In the absence of IGF-1, suspension in hyperosmotic solutions or raised hydrostatic pressure enhanced pH(i) recovery of acidified cells. This response was missing in cells incubated with IGF-1. Unresponsiveness to hyperosmotic shock represented inhibition of NHE3 activity, and was prevented using the protein kinase A inhibitor KT5720. For raised hydrostatic pressure, a decrease in NHE1 activity was responsible, and was prevented by the protein kinase C inhibitor chelerythrine.
Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl--independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl--independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl--independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl--independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl--independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.
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