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. 1991 Feb 15;274(Pt 1):15–20. doi: 10.1042/bj2740015

Measurement of intracellular Ca2+ in single aequorin-injected and suspensions of fura-2-loaded ROS 17/2.8 cells and normal human osteoblasts. Effect of parathyroid hormone.

C Schöfl 1, K S Cuthbertson 1, J A Gallagher 1, S R Pennington 1, P H Cobbold 1, G Brabant 1, R D Hesch 1, A von zur Mühlen 1
PMCID: PMC1149913  PMID: 1848074

Abstract

It is known that parathyroid hormone (PTH) activates the cyclic AMP (cAMP) signalling pathway in osteoblasts. In recent years it has been suggested that an elevation of the intracellular free Ca2+ concentration ([Ca2+]i) may also be involved in the regulation of osteoblast function by PTH. However, this remains controversial. Here we investigated the effect of PTH on the [Ca2+]i of ROS 17/2.8 cells and normal human osteoblasts. The [Ca2+]i was measured in single aequorin-injected cells and in suspensions of cells loaded with fura-2. Human PTH-(1-38)-peptide (1-300 nM) had no effect on the [Ca2+]i in single aequorin-injected ROS 17/2.8 cells (n = 17) measured at various times after injection (1-20 h), or in suspensions of fura-2-loaded ROS 17/2.8 cells (n = 9). Ionomycin (1 microM) increased the [Ca2+]i in fura-2-loaded and single aequorin-injected ROS 17/2.8 cells by 285 +/- 60 nM (n = 9) and 312 +/- 99 nM (n = 6) respectively, indicating that both methods detect changes in [Ca2+]i with equal sensitivity. In contrast, human PTH-(1-38) (10-100 nM) markedly stimulated cAMP accumulation in ROS 17/2.8 cells. In single aequorin-injected normal human osteoblasts there was no change in the [Ca2+]i in response to 100 nM human PTH-(1-38) or 100 nM bovine PTH-(1-84) (n = 18). In contrast, in suspensions of normal human osteoblasts loaded with fura-2, an increase in [Ca2+]i in response to human PTH-(1-38) (100 nM) was found (60 +/- 28 nM; n = 6). Considerable variation in the magnitude of the response was observed between individual preparations and donors. These data indicate that PTH activates cAMP accumulation without affecting [Ca2+]i in ROS 17/2.8 cells and that PTH causes a rise in [Ca2+]i only in a small subset of normal human osteoblasts. We suggest that the Ca2+ response to PTH in osteoblasts is limited by the state of differentiation of the cells, and may be due either to the presence of a distinct Ca2(+)-mobilizing receptor or to a cAMP-mediated Ca2+ response.

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Selected References

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