Microscopic properties of elementary Ca2+ release sites in nonexcitable cells

Background: Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1,4,5,-trisphosphate (InsP(3)) recep tors are the building blocks for local and global Ca2+ signalling. We previ ously found that one, or a few, Ca2+ puff sites within agonist-stimulated c ells act as 'pacemakers' to initiate global Ca2+ waves. The factors that di stinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown. Results: The spatiotemporal properties of Ca2+ puffs were investigated usin g confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ p uff sites were activated during stimulation of cells with different agonist s. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskelet on. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP(3) receptors were directly stimulated with thimerosal or membran e-permeant InsP(3) esters, Immunostaining indicated that the perinuclear po sition of pacemaker Ca2+ puffs was not due to the localised expression of I nsP(3) receptors. Conclusions: The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP(3) sen sitivity.