Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts.

Abstract

Abstract. Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H+-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60-and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatmerit of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K+-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H+-ATPase.STEOCLASTS are multinucleated giant cells that are responsible for bone resorption. Like secreting epithelial cells, they are polarized when active in bone resorption, showing three distinct specialized cell membrane areas (for review see Vaes et al., 1988). In addition to the basolateral membrane, which is rich in Na+,K+-ATPase (Baron et al., 1986), resorbing osteoclast exhibits a clear zone that mediates the attachment of resorbing cells to the bone matrix. The cytoplasm in the vicinity of this clear zone contains specialized cytoskeletal structures (Holtrop et al., 1974;Lakkakorpi et al., 1989). The third specialized membrane area, the ruffled border, faces the actual bone resorption site on the bone surface. The resorption lacuna underneath the ruffled border membrane is acidic. This has been shown by acridine orange accumulation experiments (Anderson et al., 1986; Bar6n et al., 1985) and direct micropuncture measurements (Fallon, 1984). The acidic pH favors dissolution of the bone mineral. In addition, proteinases active at acid pH and capable of collagen degradation are present in osteoclasts (Vaes et al., 1988; Blair et al., 1986). Enzyme histochemistry suggests the presenc$ of ATPase activity in the plasma membrane of the osteoclast- (Akisaka et al., 1986). Baron et al. (1985) found at the ruffled:border of the osteoclast a 100-kD lysosomal membrane polypeptide that showed immunological similarity to gastric H+,K+-ATPase. Now we report that osteoclasts contain an ATP-dependent proton pump that is clearly different from the gastric proton pump and from the mitochondrial proton pump but shows considerable immunological similarities to the vacuolar type H+-ATPase of Neurospora crassa. Materials and Methods Preparation of Bone MicrosomesBone microsomes were prepared from medullary bone of regularly laying hens. Medullary bone from the tibia and femur was dissected out and immediately homogenized in a medium containing 5 mM Tris pH 7.4, 250 mM sucrose, 1 mM K2CO3, 1 mM DTT, and 1 mM EGTA in a glass-Teflun ...

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity

“…Furthermore, there is a positive correlation between metastatic potential and the level of V-ATPase activity at the plasma membrane (26), suggesting that V-ATPases provide the acidic extracellular environment necessary for invasion. V-ATPases have also been shown to be localized to the plasma membrane of various specialized cells, such as osteoclast (54), neutrophils (14,32,34), kidney intercalated cells (9,13,47), the mitochondria-rich cells of the rat epididymis (4), and in angiogenic cells (41).…”

“…V-ATPase is a member of a family of ATP-driven proton pumps responsible for the acidification of intracellular compartments such as endosomes, lysosomes, Golgi-derived vesicles, and clathrin-coated vesicles (35). In addition to the role of V-ATPase in intracellular compartments, this enzyme is important for plasma membrane functions in various specialized cells (13,32,54).There are several classes of inhibitors of V-ATPase, including the macrocyclic lactones bafilomycin and concanamycin, the benzolactone enamides salicylihalamides and lobotamides, and, more recently, the macrolide lactams chondropsin and poecillastrin (3). Although they exhibit different potencies and selectivity to inhibit V-ATPases from mammals and fungal sources, they all seem to bind to subunit c to exert their effect.…”

“…V-ATPase is a member of a family of ATP-driven proton pumps responsible for the acidification of intracellular compartments such as endosomes, lysosomes, Golgi-derived vesicles, and clathrin-coated vesicles (35). In addition to the role of V-ATPase in intracellular compartments, this enzyme is important for plasma membrane functions in various specialized cells (13,32,54).…”

A cytochemical assay for osteoclast cathepsin K activity

“…The resorbing osteoclast creates an extracellular compartment which it maintains at an acid pH via a vacuolar H þ -ATPase, and into which it secretes hydrolytic enzymes. [1][2][3][4][5] The acidity of the compartment demineralizes the matrix, and provides an optimal pH for proteolytic enzymes which degrade the demineralized matrix. A convoluted membrane, the ruffled border, 4 lines the resorption lacuna that forms beneath an attached osteoclast.…”

“…[1][2][3][4][5] The acidity of the compartment demineralizes the matrix, and provides an optimal pH for proteolytic enzymes which degrade the demineralized matrix. A convoluted membrane, the ruffled border, 4 lines the resorption lacuna that forms beneath an attached osteoclast. Lysosomal enzymes, packaged into transport vesicles, are transported to the ruffled border, where they are secreted into the underlying compartment.…”