The subcellular distribution of [¹⁴C]dichloromethylenebisphosphonate and [¹⁴C]1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells

Summary

Rat calvaria cells were cultured for 6 days in the presence or absence of [¹⁴C]dichloromethylenebisphosphonate ([¹⁴C]Cl₂MBP) or [¹⁴C]1-hydroxyethylidene-1, 1-bisphosphonate ([¹⁴C]HEBP), after which cell organelles were separated by differential centrifugation. The distribution of protein, glutamate dehydrogenase, acid phosphatase, and 5'-nucleotidase was similar for cells treated or not treated with Cl₂MBP. About 70–80% of the [¹⁴C]Cl₂MBP and [¹⁴C]HEBP was found to be present in the supernatant. This was the only fraction that showed a ratio higher than 1 for the relative specific radioactivity, indicating that the bisphosphonates accumulated mainly in the cytosol. Rapid separation of particulate components and soluble cytoplasm of cells treated with [¹⁴C]Cl₂MBP confirmed this finding, showing that it is unlikely that the result was due to leakage from the organelles. The uptake of [¹⁴C]Cl₂MBP into cells was similar in different cell types. The binding of both bisphosphonates to macromolecules in the medium was 0.1–0.2% and 1–4% in the cells. This binding is not due to metabolic activity of the cells. About 15–20% of [¹⁴C]HEBP and [¹⁴C]Cl₂MBP was modified by the living cells.