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.