Ultrastructural immunocytochemical localization of carbonic anhydrase in normal and calcitonin‐treated chick osteoclasts

Abstract

Carbonic anhydrase was localized in osteoclasts by light and electron microscopy using a preembedding peroxidase-antiperoxidase staining method. Osteoclasts on the endosteal surface of the metatarsi of calcitonin-treated and untreated (control) chicks were studied. A positive staining reaction was seen in the cytosol, in the Golgi apparatus, in and on vesicles, on the plasma membrane of the ruffled border, and on the bone surface beneath control osteoclasts. After calcitonin treatment, positive staining reactions were seen at the same sites except that staining was absent on the plasma membrane and endosteal bone surface. The morphology of osteoclasts from calcitonin-treated chicks was indicative of cell inactivity. The carbonic anhydrase which was bound to the plasma membrane of the ruffled border is appropriately arrayed for hydrogen ion secretion and subsequent mineral dissolution. The presence of the enzyme within lysosomelike vesicles and on the endosteal surface beneath active cells suggests that it may be released into the resorbing zone along with the lysosomal hydrolases. The function of the extracellular enzyme is also unknown.

Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.

“…As in these ceils (17), reversible vesicular insertion of proton pumps at the secretory pole might modulate acid secretion by osteoclasts (4). Second, acid-secreting gastric and urinary cells also contain numerous mitochondria and very high carbonic anhydrase activity in the cytosol (1,26) as does the osteoclast (2,14,15,43). Inhibition of carbonic anhydrase activity prevents bone resorption in organ culture (28) and affects calcium metabolism in the intact animal (46).…”

“…× 9,000. tion areas was regarded as indicative of a low pH at these sites (31). Third, carbonic anhydrase, an enzyme associated with several acid-secreting epithelia, was demonstrated to be involved in bone resorption (28,46) and to be specifically concentrated in osteoclasts (2,14,15,43). Despite these suggestions, direct evidence of a low pH at the bone-resorbing lacuna underlying the osteoclast is still lacking and the acidification mechanisms are not yet elucidated.…”

1 alpha,25-dihydroxyvitamin D3 regulates the transcription of carbonic anhydrase II mRNA in avian myelomonocytes.

“…The cells were suspended in 10 mM Tris-HCl, pH 7.4/10 mM NaCl/3 mM MgCl2/0.5% Nonidet P-40 and disrupted by repeated pipetting through a Pasteur pipet. After centrifugation, the nuclei (2-3 x 107) were suspended in 200 ,ul of a transcription buffer containing 40%(vol/vol) glycerol, 10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 2 This procedure yields labeled RNA with a total activity of 1-2 x 107 cpm/assay. Equal numbers of counts (107 cpm per reaction) from 1,25(OH)2D3-treated and control BM2 cells were hybridized to denatured cDNAs applied to a nylon membrane (Hybond-N+).…”

“…The carbonic anhydrase isoform II (CAII) is highly expressed in cells where acid-base regulation is a primary function-i.e., gastric parietal cells, salivary glands, renal tubular cells (1), and the osteoclast in bone (2,3). In all these cells, the role of CAII is to generate H+ for acid extrusion by the ATP-driven proton pumps, and HCO-is usually extruded via bicarbonate/chloride exchangers.…”

Molecular Understanding of Osteoclast Differentiation and Physiology

“…Several enzymes including carbonic anhydrase II supply protons for the proton pump [19]. In resorbing osteoclasts, carbonic anhydrase II is highly expressed, whereas in nonresorbing osteoclasts only low basal expression is maintained.…”