S-PHASE INDEPENDENCE OF PARATHYROID HORMONE-INDUCED CALCIUM SIGNALLING IN PRIMARY OSTEOBLAST-LIKE CELLS

Abstract Cell cycle-dependence of parathyroid hormone (PTH)-induced signal transduction was studied at the single cell level in fetal rat osteoblast-like cells (ROB) in primary culture. Responsiveness to 10 −7 M rPTH1–34 was measured as changes in the intracellular calcium concentration ([Ca 2+ ] i ) of cells loaded with Fura-2 using a video imaging setup. Cells in S-phase were identified by staining for incorporated 5-bromo-2′-deoxyuridine (BrdU). ROB were cultured on glass coverslips which had alphanumerically marked gratings so that cells could be located. Directly after measurement of the PTH-induced [Ca 2+ ] i response of the individual cells, cells were fixed and stained for incorporated BrdU. Phasecontrast images taken with the video imaging setup were compared with phase-contrast micrographs and fluoro-micrographs taken after staining for BrdU incorporation. We found that 43% of the cells responding to PTH with a rise in [Ca 2+ ] i had also incorporated BrdU. This percentage was not different from the percentage BrdU positive cells in the whole culture, showing that calcium responsiveness was randomly distributed between ROB in S-phase and those not in S-phase. We therefore conclude that PTH does not induce calcium responses preferentially during the S-phase of the cell cycle in ROB in primary culture.