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The effects of parathyroid hormone (PTH) on 1,4,5‐inositol triphosphate (1,4,5‐IP3) and intracellular free calcium (Cai2+) 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 Cai2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH‐stimulated Cai2+ 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 Cai2+ changes. In suspended cells loaded with the calcium indicator dye fura‐2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H‐7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Cai2+ (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 Cai2+ 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 (∼ λ510 nm) detected and optimized at λ348/374 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 Cai2+ 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 Cai2+ 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 × 30 min). PTH‐induced Cai2+ increases in adherent PKC inhibitor‐treated cells were also associated with a 1.8‐fold 1,4,5‐IP3 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‐IP3/Cai2+). J. Cell. Biochem. 65:276–285. © 1997 Wiley‐Liss, Inc.
Journal of Cellular Biochemistry – Wiley
Published: May 1, 1997
Keywords: ; ; ; ; ;
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