Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells
✍ Scribed by Michael Babich; Lisa R.P. Foti; Kevin L. Mathias
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 388 KB
- Volume
- 65
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP 3 ) and intracellular free calcium (Ca i 21 ) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Ca i 21 remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca i 21 increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Ca i 21 changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM 3 1 h) and H-7 (30 µM 3 18 h) potentiated the effects of 1 µg/ml bPTH(1-84) on Ca i 21 (83% increase over basal) by 1.4-and 1.65-fold, respectively. In comparison, PTH (10 ng-1 µg/ml) was without significant effect on adherent cell Ca i 21 as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (,l510 nm) detected and optimized at l348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Ca i 21 increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Ca i 21 in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 µM 3 30 min). PTH-induced Ca i 21 increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP 3 increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP 3 /Ca i 21 ).