Parathyroid hormone (PTH) stimulates cytosolic signalling pathways in human osteoblasts, although the precise pathways have not been determined. Previous experiments suggest that PTH causes increases in cell volume in osteoblasts. The current series of experiments were designed to further elucidate the signalling pathway.
Transformed human osteoblasts, derived from an osteogenic sarcoma cell line (Saos-2), were cultured in alpha modification Dulbecco’s modified Eagle’s medium (DMEM), with 10 % heat-inactivated fetal calf serum, 100 U ml-1 penicillin, 10 µg ml-1 streptomycin, 2.5 µg ml-1 amphotericin B and 4 mM L-glutamine at 37 °C in a humidified atmosphere with 5 % carbon dioxide.
Cells (2 Ω 104 ml-1) were seeded on to Pioloform covered gold electron microscopy grids contained in a 4-well tissue culture plate. Six hours prior to stimulation the culture medium was removed and replaced with DMEM containing 250 ng ml-1 cholera toxin (CTX). After incubation the cells were exposed to 2.5 µM PTH for 0, 5, 15, 30 and 60 s. They were then washed in ice-cold distilled water for 10 s; excess water was removed by blotting, and the cells cryofixed by immersion in liquid nitrogen (-210 °C). The cells were freeze dried and coated with carbon.
Areas of cytoplasm and nucleus (1 Ω 2.5 µm) were analysed for 60 s live-time using a Zeiss electron microscope with a Link AN 10 000 microanalysis system. Spectra were quantified by the continuum normalisation technique using gelatin standards (Warley, 1997). Data were analysed by the Mann-Whitney test; differences were considered significant for P < 0.05.
Cells cultured in PTH showed a generalised decrease in the concentrations of Na+ and K+ within the cytoplasm after 30 s, which recovered towards control values by 60 s (Table 1). In cells cultured with PTH + CTX, concentrations of Na+ and K+ decreased after 30 s and remained low (Table 1).
Element concentration changes induced by PTH appear to be linked to G protein activation and can be modified by CTX.
- Warley, A. (1997). X-Ray Microanalysis for Biologists, vol. 16, in the series Practical Methods in Electron Microscopy, series ed. Glauert, A.M. Portland Press, London.