Tattersall Amanda, Meredith David, Furla Paola, Shen Meng-Ru, Ellory Clive, Wilkins Robert
Department of Human Anatomy and Genetics, South Parks Road, Oxford.
Cell Physiol Biochem. 2003;13(4):215-22. doi: 10.1159/000072424.
Cartilage matrix turnover is sensitive to changes in intracellular pH (pH(i)) and previous studies have shown that articular chondrocytes regulate pH(i) 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 pH(i) 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.
软骨基质更新对细胞内pH值（pH(i)）的变化很敏感，先前的研究表明，关节软骨细胞主要通过一种对阿米洛利敏感的Na(+) / H(+)交换体（NHE）来调节pH(i)，该交换体具有管家亚型NHE1的典型特性。然而，免疫印迹研究表明，牛软骨细胞表达Na(+) / H(+)交换体的NHE1和NHE3两种亚型。在本研究中，研究了血清暴露对软骨细胞酸排出的影响。使用对pH敏感的荧光探针BCECF来测量分离的牛关节软骨细胞中的pH(i)，并对在无5%胎牛血清（FBS）和有5%胎牛血清存在的情况下分离的细胞的质子等效膜转运体进行了表征。在铵诱导酸化后，使用离子替代和特异性NHE1抑制剂HOE694的组合来评估NHE亚型对酸排出的贡献。虽然在未用FBS处理的细胞中，HOE694完全抑制了依赖Na(+)的酸排出，但在暴露于FBS的细胞中检测到了NHE1和NHE3的活性。同时，RT-PCR和免疫组织化学实验分别证实了NHE1和NHE3的mRNA以及这两种蛋白质的表达。虽然血清生长因子实际上不会进入健康软骨，但它们可以渗透受损的基质。NHE3的调节特性与NHE1不同，因此对机械应力成分（如高渗）的反应模式改变可能与疾病组织中生长因子的进入增加有关。
