Intracellular calcium puffs in osteoclasts

We studied intracellular calcium ([Ca2+](i)) in acid-secreting bone-attache d osteoclasts, which produce a high-calcium acidic extracellular compartmen t. Acid secretion and [Ca2+](i) were followed using H+-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was simi lar to 100 nM, similar to cells on inert substrate that do not secrete acid . However, measurements in restricted areas of the cell showed [Ca2+](i) tr ansients to 500-1000 nM consistent with calcium puffs, transient (milliseco nd) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. T ransients did not propagate in waves across the cell in scanning confocal m easurements. Calcium puffs occurred mainly over regions of acid secretion a s determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-nega tive nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-su rface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca2+](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca2+](i) transients in o steoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transp ort requires calcium, suggesting that calcium puffs function to maintain ac id secretion. However, membrane H+-ATPase activity was insensitive to calci um in the 100 nM-1 mu M range. Thus, any effects of calcium puffs on osteoc lastic acid transport must be indirect. (C) 1999 Academic Press.