EFFECTS OF CELL-CULTURE TIME AND BONE-MATRIX EXPOSURE ON CALMODULIN CONTENT AND ATP-DEPENDENT CELL-MEMBRANE ACID TRANSPORT IN AVIAN OSTEOCLASTS AND MACROPHAGES

Osteoclasts mediate bone resorption by secretion at the site of bone a ttachment. This process depends on calmodulin concentrated at a specia lized acid-secreting membrane. We hypothesized that increased calmodul in and bone attachment were required for acid secretion. We tested thi s by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells. Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone. Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake. Calmodulin decreased in osteoclasts cultured without bone. Calmodulin in isolated macrophages was similar to 25% of osteoclast levels, but increased several fold by 5 days. Bo ne had no effect. Calmodulin mRNA was similar in osteoclasts with or w ithout bone. However, only osteoclasts cultured with bone retained aci d transport capacity. Macrophage calmodulin mRNA was not affected by b one, but increased three fold by day 5, paralleling protein production . Macrophages developed acid transport capacity at 3-5 days, but at lo wer levels than osteoclasts, and bone had no measurable effect. Chicke n cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We c onclude that, following isolation, calmodulin levels decline in osteoc lasts via a post-transcriptional mechanism. In cultured macrophages, b y contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentia tion in vitro. Increased calmodulin correlated with membrane acid tran sport capacity in both cell types. The macrophage findings indicate th at stimuli other than bone influence acid transport capacity in this f amily of cells. (C) 1996 Wiley-Liss, Inc.