title: Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones.
abstract: Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.
mesh_terms: D000634:Aminoquinolines; D000818:Animals; D001862:Bone Resorption / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015232:Dinoprostone; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin; D008564:Membrane Potentials; D051379:Mice; D012516:Osteosarcoma / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D011458:Prostaglandins E / Q000494:pharmacology; D051381:Rats; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; D004987:Ethers; D010281:Parathyroid Hormone; D011458:Prostaglandins E; D014660:Vasoactive Intestinal Peptide; D015759:Ionomycin; D004815:Epidermal Growth Factor; C035738:Quin2-acetoxymethyl ester; D000242:Cyclic AMP; D015232:Dinoprostone; D002118:Calcium