Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells.
We previously demonstrated that the [Ca2+]i 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 FlCRhR. FlCRhR 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 FlCRhR 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.
Civitelli
R
R
Division of Bone and Mineral Diseases, Washington University School of Medicine, Jewish Hospital of St. Louis, Missouri.
Bacskai
B J
BJ
Mahaut-Smith
M P
MP
Adams
S R
SR
Avioli
L V
LV
Tsien
R Y
RY
D000818:Animals; D001859:Bone Neoplasms / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000187:drug effects*; D012516:Osteosarcoma / Q000473:pathology; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D019379:Teriparatide; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; C058350:parathyroid hormone (7-34); D019379:Teriparatide; C031617:parathyroid hormone (3-34); D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium