DETECTION OF A TRIGGER ZONE OF BRADYKININ-INDUCED FAST CALCIUM WAVES IN PC12 NEURITES

Bradykinin and caffeine were used as two different agonists to study i nositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sen sitive intracellular Ca2+ release in the outgrowing neurites of nerve- growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Change s in neuritic intracellular free Ca2+ ([Ca2+](i)) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) e ster of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination wi th confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ r elease was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentration s of bradykinin (>500 nM) arose from a trigger zone in the proximal pa rt of the neurite, as a bi-directional wave towards the growth cone an d cell body. The speed of neuritic Ca2+ waves was reduced in cells loa ded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethanetetraacetic ac id/AM. Preloading of Ca2+ stores led to increased bradykinin-induced C a2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffei ne evoked a simultaneous [Ca2+](i) rise along the neurites of Ca2+ pre loaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speed s, but no longer-lasting IP3-induced [Ca2+](i) signals, correlated wit h increased caffeine-induced Ca2+ release in the neurites. At low conc entrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propaga te as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs sprea d across only a few microns, at a slower speed than neuritic Ca2+ wave s. These Ca2+ puffs represent elementary Ca2+ release units, whereby t he released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.