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.

Parathyroid Hormone Potentiates Nucleotide-induced [Ca2+] Release in Rat Osteoblasts Independently of Gq Activation or Cyclic Monophosphate Accumulation

“…This is in contradiction to Kaplan et al (17) who reported that PTH did induce a small [Ca 2ϩ ] i elevation. Earlier studies also confirm that the effect of PTH on [Ca 2ϩ ] i mobilization in osteoblasts is controversial, one group finding no effect (24), but others reporting various increases (25,26). A possible explanation for these very different reports may be the presence of ATP in the culture medium due to cell lysis.…”

A New Action of Parathyroid Hormone

“…Previous studies on SaOS-2 cells reported that PTH did not stimulate acute increases in [Ca 2ϩ ] i (33). However, PTH has been shown to elicit transient increases in [Ca 2ϩ ] i and generate inositol polyphosphates in other osteoblast-like cell lines (4,5,15); therefore, we examined the role of the inositol lipid/Ca 2ϩ / PKC signaling pathway in the stimulation of ECAR by PTH.…”

“…A minimum of five consecutive data points from the base-line ECAR of each cell population were selected and averaged, and then all other data points were expressed as a percentage of this basal average. (32,33). In brief, the cells in the monolayer were detached from the culture dish using Ca 2ϩ -free Hanks' balanced salt solution (HBSS) consisting of 120 mM NaCl, 4 mM KCl, 10 mM D-glucose, and 20 mM HEPES, pH 7.2, supplemented with 5 mM EDTA.…”

“…sponse to PTH using single wavelength fluorescence monitoring (33). However, in other osteoblastic cell strains, PTHR occupancy has been linked to activation of the inositol lipid signaling pathway (4,5,15).…”

Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase.

“…This cDNA probably represents ALP of bone origin since it was derived from Saos-2 osteosarcoma cells that have osteoblastic properties (7). Partial amino acid sequences obtained from purified liver ALP are identical to portions of the polypeptide encoded by the L/B/K cDNA.…”