SINGLE-CELL ANALYSIS OF CYCLIC-AMP RESPONSE TO PARATHYROID-HORMONE INOSTEOBLASTIC CELLS

We previously demonstrated that the [Ca2+](i) response to PTH is heter ogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whet her response heterogeneity is a universal feature of PTH signal transd uction, cAMP production was monitored in monolayer cultures of UMR-106 -01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-s ensitive fluorescent indicator FICRhR. FICRhR was microinjected into s ingle cells, and the 500-530/>560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentra tion ([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 elev ated for at least 30 minutes. This effect was accompanied by a slow tr anslocation of the fluorescein-labeled catalytic subunit of protein ki nase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) cause d no detectable increase in [cAMP] in HOB cells, although PGE(2) (3 x 10(-6) M) stimulation was able to increase the FICRhR ratio (154 +/- 2 7%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent a t PTH(1-34), but deletion of the first two amino acids at the N termin us 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 ind uced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells s timulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+](i) response.