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 inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.
Reber
B F
BF
Department of Pharmacology, University of Bern, Friedbuehlstrasse 49, CH-3010 Bern, Switzerland.
Schindelholz
B
B
D000814:Aniline Compounds; D000818:Animals; D001572:Benzofurans; D001920:Bradykinin / Q000494:pharmacology*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005071:Evoked Potentials / Q000187:drug effects; D007093:Imidazoles; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D018613:Microscopy, Confocal; D009414:Nerve Growth Factors / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D012433:Ryanodine / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D001572:Benzofurans; D007093:Imidazoles; D009414:Nerve Growth Factors; D014966:Xanthenes; C082877:fura red; D012433:Ryanodine; C059715:Fluo-3; D002110:Caffeine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D001920:Bradykinin; D002118:Calcium