In Vitro Effects of Ionophore A23187 on Skeletal Collagen and Noncollagen Protein Synthesis*

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Abstract
Effects of ionophore A23187 on skeletal collagen formation were investigated in vitro. Collagen synthesis was quantitated in fetal rat calvaria by measuring [3H]proline incorporation into collagenase-digestible (CDP) and noncollagen protein (NCP) using purified bacterial collagenase; [3H]proline was added for the last 2 h of culture. Results are as follows. 1) A23187 (0.03–1.0 jug:/ml) inhibited incorporation of label into CDP and NCP after 24 h of culture, with a greater effect on CDP. The response was not associated with altered amino acid uptake, precursor pool size, or degradation of newly labeled protein. 2) Submaximal concentrations of A23187 and parathyroid hormone or dibutyryl cAMP decreased CDP formation to a greater extent than treatment with the agents alone. 3) Imidazole, while ineffective by itself, enhanced the effect of A23187. 4) Alteration of medium calcium did not affect the response to ionophore. 5) The inhibitory effect of A23187 was partially reversed by 24 h and completely reversed by 48 h of control treatment subsequent to an initial 24-h incubation with ionophore. 6) Indomethacin had no effect on CDP or NCP formation, either in the presence or absence of A23187. 7) A23187 did not alter the uptake of [3H]thymidine or [3H]uridine into acid-extractable pools but decreased incorporation of label into DNA and RNA, respectively. 8) Histological examination showed no difference between control and A23187 treatment after 24 h. We conclude that A23187 decreases bone collagen and noncollagen protein synthesis, possibly through a calcium-mediated effect. The mechanism of the inhibitory effect on DNA and RNA labeling is unknown, although it may be related to calcium. Our results further suggest that calcium may be involved in the actions of parathyroid hormone and dibutyryl cAMP on skeletal collagen synthesis.