Calcium Puffs Research Articles

Intracellular Calcium Puffs in Osteoclasts

We studied intracellular calcium ([Ca2+]i) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. 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 ∼100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca2+]i transients to 500–1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5–15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface 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 osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H+-ATPase activity was insensitive to calcium in the 100 nM–1 μM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.

From calcium blips to calcium puffs: theoretical analysis of the requirements for interchannel communication.

In the cytoplasm of cells of different types, discrete clusters of inositol 1,4,5-trisphosphate-sensitive Ca(2+) channels generate Ca(2+) signals of graded size, ranging from blips, which involve the opening of only one channel, to moderately larger puffs, which result from the concerted opening of a few channels in the same cluster. These channel clusters are of unknown size or geometrical characteristics. The aim of this study was to estimate the number of channels and the interchannel distance within such a cluster. Because these characteristics are not attainable experimentally, we performed computer stochastic simulations of Ca(2+) release events. We conclude that, to ensure efficient interchannel communication, as experimentally observed, a typical cluster should contain two or three tens of inositol 1,4,5-trisphosphate-sensitive Ca(2+) channels in close contact.

Calcium signals in the cell nucleus. Strasbourg, France, August 20-23, 1998.

Just as the great explorations revealed a world far more diverse and rich than had been imagined before, a mass of fast accumulating data is disclosing a much more complex and sophisticated picture of the nucleus than hitherto assumed. The nucleus has long been thought of as some sort of enclosure circumscribed by a double membrane and containing the genetic material of the cell. The inner and outer nuclear membranes join periodically at the nuclear pores, forming complexes supposed to create large channels for the free diffusion of ions and small macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted, with nuclear Ca2+ signals originating from cytosolic Ca2+ waves merely by passive transmission through the nuclear pore complex (NPC). However, recent evidence has changed the mappa mundi of the cell. It has been shown that nuclear Ca2+ can be regulated independently of cytosolic Ca2+ changes. And, just as the Terra Incognita discovered beyond the Ocean Sea proved a fabulous New World, the nuclear envelope (NE) between the cytoplasm and nucleoplasm appears to play an essential role in the regulation of Ca2+ signals inside the nucleus. It contains proteins that regulate and respond to changes in nucleosolic Ca2+ concentration ([Ca2+]nucleosol). Furthermore, several specific Ca2+‐dependent nuclear functions have been described and the list is growing. The regulation of nuclear Ca2+ signals, and the role of nuclear Ca2+ as a specific regulator of nuclear events through Ca2+‐binding proteins, were the main themes of the recent EMBO workshop entitled ‘Calcium signals in the cell nucleus’, organized by A.N.Malviya and coworkers in Strasbourg, France. The existence of Ca2+ signals at the level of the nucleoplasm has never been a real issue, as the …

Activation and co-ordination of InsP3-mediated elementary Ca2+ events during global Ca2+ signals in Xenopus oocytes.

1. The activation of elementary calcium release events ('puffs') and their co-ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. 2. Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near-simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3]. 3. Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co-operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback. 4. The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly. 5. Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non-constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium. 6. Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10-fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies. 7. We conclude that the initiation of calcium liberation depends co-operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.

Nuclear calcium signalling by individual cytoplasmic calcium puffs.

It is known that the nucleoplasmic ionised calcium concentration (Can) controls nuclear functions such as transcription, although the source and nature of the signals which modulate Can are unclear. Using confocal imaging, we investigated the subcellular origin of Can signals in Fluo-3-loaded HeLa cells. Our data indicate that all signals which increased Can were of cytoplasmic origin. Can was elevated during the propagation of global Ca waves within cells. More strikingly, we found that individual cytoplasmic elementary release events e.g. Ca puffs, evoked by physiological levels of stimulation, caused transient Can increases. Significantly, >70% of all Ca puffs originated within a 2-3 micron perinuclear zone and propagated anisotropically across the entire nucleus. Due to the relatively slow relaxation of Can transients compared with those in the cytoplasm, repetitive perinuclear Ca puffs were integrated into a 'staircase' of increasing Can. Due to the effective diffusion of Ca in the nucleoplasm, the nucleus served as a 'Ca tunnel', distributing Ca to parts of the cytosol which were otherwise not within the cytoplasmic diffusion radii of Ca puffs. Given the close proximity of the majority of puff sites to the nucleus, it seems that the elementary Ca release system is designed to facilitate nuclear Ca signalling. Consequently, Ca-dependent regulation of nuclear function must be considered at the microscopic elementary level.