title: 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.
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.
authors.affiliation: Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover, Federal Republic of Germany.
authors.forename: C
authors.initials: C
authors.lastname: Schöfl
authors.forename: K S
authors.initials: KS
authors.lastname: Cuthbertson
authors.forename: J A
authors.initials: JA
authors.lastname: Gallagher
authors.forename: S R
authors.initials: SR
authors.lastname: Pennington
authors.forename: P H
authors.initials: PH
authors.lastname: Cobbold
authors.forename: G
authors.initials: G
authors.lastname: Brabant
authors.forename: R D
authors.initials: RD
authors.lastname: Hesch
authors.forename: A
authors.initials: A
authors.lastname: von zur Mühlen
mesh_terms: D000331:Aequorin; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D016257:Fura-2; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*
chemical_list: D010281:Parathyroid Hormone; D005576:Colforsin; D000001:Calcimycin; D000331:Aequorin; D015759:Ionomycin; D000242:Cyclic AMP; D002118:Calcium; D016257:Fura-2