The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cellsubstratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and o~-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and a-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclastspecific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.Osteoclasts are specialized cells involved in bone resorption. Even if many aspects of their function in bone physiology are not yet known, their morphology and ultrastructure are known in some detail (1,15,18). In particular, a ruffled border has been identified and described by both electron microscopy and microcinematography as a highly motile peripheral area probably involved primarily in bone resorption (17). An organelle-free area called "clear zone" or "sealing zone" (18) immediately adjacent to the ruffled border has been described. Schenk et al. (28) have suggested that the clear zone provides the osteoclast with a tight bone attachment area; King and Holtrop (21) have shown, in this same area, organized bundles of actin filaments ending within short processes contacting the bone surface. Further evidence that the clear zone is a specialized site of attachment has been provided also in different experimental conditions (27,30).Recently, a technique for isolating and culturing functional osteoclasts free of other cell types has been developed (36, 39); using this isolation procedure, the cytoskeleton of osteoclasts has been studied in vitro by means of immunofluorescence microscopy (37). A major finding was that in most osteoclasts, and especially in those highly flattened, multiple actin-containing dots were located in the area that topographically and ultrastructuraUy corresponds to the clear zone observed in vivo. In this same area, dot-like points of close adhesion and a belt of intermediate filaments of the vimentin type were also located, while fibronectin was excluded from the whole attachment area (37). Having in mind the idea that the whole idea corresponding to the clear zone could represent a...
Abstract. The mechanisms of Ca 2÷ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltagegated Ca 2+ channels (VGCC) were detected by the effects of membrane depolarization with K ÷, BAY K 8644, and dihydropyridine antagonists. K + produced dose-dependent increases of cytosolic calcium ([Ca2+]~) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K +. The effects of K + were completely inhibited by dihydropyridine derivative Ca 2+ channel blocking agents. BAY K 8644 (5 x 10 -6 M), a VGCC agonist, stimulated Ca 2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca 2+ entry mechanisms as- BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.
Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagendegrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption.Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [CI-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.
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