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 attachment. This process depends on calmodulin concentrated at a specialized acid-secreting membrane. We hypothesized that increased calmodulin and bone attachment were required for acid secretion. We tested this 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 approximately 25% of osteoclast levels, but increased several fold by 5 days. Bone had no effect. Calmodulin mRNA was similar in osteoclasts with or without bone. However, only osteoclasts cultured with bone retained acid transport capacity. Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production. Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect. Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism. In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro. Increased calmodulin correlated with membrane acid transport capacity in both cell types. The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.