Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization.
The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH(3) cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.
Santillo
Mariarosaria
M
Department of Neuroscience, Section of Physiology, University of Naples Federico II, Naples, Italy.
Secondo
Agnese
A
Serù
Rosalba
R
Damiano
Simona
S
Garbi
Corrado
C
Taverna
Elena
E
Rosa
Patrizia
P
Giovedì
Silvia
S
Benfenati
Fabio
F
Mondola
Paolo
P
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000201:enzymology* / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D013482:Superoxide Dismutase / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D013574:Synaptosomes / Q000737:chemistry / Q000201:enzymology*
D015220:Calcium Channels; D009498:Neurotoxins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D019274:Botulinum Toxins, Type A; D011188:Potassium; D002118:Calcium