Molecular and functional identification of the Na(+)/H(+) exchange isoforms NHE1 and NHE3 in isolated bovine...

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.