Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells

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Abstract
We previously demonstrated that the [Ca2+], response to PTH is heterogeneous in single UMR‐106‐01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR‐106‐01 cells and human trabecular bone osteoblasts (HOB) using the cAMP‐sensitive fluorescent indicator FICRhR. FICRhR was microinjected into single cells, and the 500‐530/>560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR‐106‐01 cells exposed to bovine PTH(1‐34) (10−7 M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 + 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10−7 M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein‐labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1‐34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10−6 M) stimulation was able to increase the FICRhR ratio (154 + 27%, n = 10). The truncated fragment PTH (2‐34) was only 67% as potent at PTH(1‐34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR‐106‐01 cells. Brief exposure to 10−7 M of either PTH(3‐34) or PTH(7‐34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1‐34). Thus, in osteoblast‐like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.
Funding Information
  • NIH (AR 32087 (Civitelli and Avioli), AR 41255 (Civitelli), NS27177 (Tsien))
  • NIH Training (AR07033 (Avioli))