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.