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Protein kinase C modulator effects on parathyroid hormone‐induced intracellular calcium and morphologic changes in UMR 106‐H5 osteoblastic cells
Author(s) -
Babich Michael,
Foti Lisa R.P.,
Mathias Kevin L.
Publication year - 1997
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(199705)65:2<276::aid-jcb13>3.0.co;2-g
Subject(s) - parathyroid hormone , intracellular , calcium in biology , calcium , chemistry , endocrinology , microbiology and biotechnology , medicine , protein kinase c , kinase , biology , biochemistry , organic chemistry
The effects of parathyroid hormone (PTH) on 1,4,5‐inositol triphosphate (1,4,5‐IP 3 ) and intracellular free calcium (Ca i 2+ ) 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 2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH‐stimulated Ca i 2+ 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 2+ 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 Ca i 2+ (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 2+ 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 Ca i 2+ 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 2+ 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 Ca i 2+ 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 2+ ). J. Cell. Biochem. 65:276–285. © 1997 Wiley‐Liss, Inc.