We have previously demonstrated by immuno-histochemical analysis that the α, β and γ subunits of ENaC are expressed in human chondrocyte-like cells (Mobasheri et al. 1999) and that the expression of ENaC subunits in chondrocytes is sensitive to extracellular [Na+] (Trujillo et al. 2000). Since ENaC has been proposed to function as a non-selective, mechanosensitive cation channel (Kizer et al. 1997; Mobasheri & MartÆn-Vasallo, 1999), in this study, we examined the expression of ENaC subunits in related human skeletal cells including bone-derived osteoblasts and human anterior cruciate ligament (ACL) cells.
Human bone-derived osteoblasts were isolated from trabecular bone chips essentially as previously described (Gundle & Beresford, 1995). Human ACL cells were obtained from anterior cruciate ligament during elective surgery from knee replacement operations with approval of the patient and the Hospital Medical Staff Ethics Committee. For immunofluorescence, cells were cultured on circular glass coverslips in DMEM supplemented with 10 mM NaHCO3, 25 mM Hepes, 2 mM L-glutamine, 10 % FCS, 30 units ml-1 benzylpenicillin and 15 µg ml-1 streptomycin sulphate, 100 nM L-ascorbic acid-2-phosphate and 10 nM dexamethasone (Fermor et al. 1998). The cells were fixed in freshly prepared 3.7 % paraformaldehyde in PBS for 10 min, blocked with 10 % normal goat serum and probed with polyclonal antibodies raised against the α, β and γ subunits of ENaC and secondary TRITC-conjugated anti-rabbit antibodies. The immunostained cells were examined using a Leica confocal laser scanning microscope. Human specific oligonucleotide primers designed to amplify the α, β and γ subunits of ENaC were used to detect transcripts of ENaC in cDNA libraries prepared from human osteoblasts and human ACL cells.
Immunofluorescence confocal microscopy revealed moderate to low levels of αENaC expression in human osteoblasts and human ACL cells compared with human chondrocytes. RT-PCR confirmed these findings; low levels of αENaC expression were detected in human osteoblasts and human ACL cells (Fig. 1) but no evidence could be found for expression of the β or γ subunits of ENaC.
This study demonstrates that only the α subunit of ENaC is expressed in human osteoblasts and human ACL cells. These results confirm earlier observations made in osteoblasts (Kizer et al. 1997) and provide further evidence for the expression of αENaC in skeletal cell types. The absence of β and γ ENaC suggest that αENaC may be implicated as a multipurpose ion channel capable of transducing mechanical signals in skeletal cells for such purposes as initiating volume regulatory responses and sensing mechanical load through transduction of mechanical signals.
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| Figure 1. RT-PCR evidence for αENaC expression in human osteoblasts and human ACL cells. The faint 600 base pair products correspond to αENaC, which was found to be expressed in both human osteoblasts and ACL cells. β-Actin (310 base pairs) was used as an internal control. |
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