Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion

Abstract

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca²⁺] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca²⁺, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca²⁺]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 μM and 90% at 7 μM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 μM trifluoperazine. Quantitative immunofluorescence using fluorescein-labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3-day bone-attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP-dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent K_m for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated.