Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons
Section snippets
Determination of sPLA2 activity of crotoxin subunit B and purified human synovial sPLA2
The activity of the two sources of sPLA2 used in this study was determined as previously described (Thwin et al., 2002). In brief, [3H]arachidonate-labeled E. coli membrane suspension (0.005 mCi/ml; 5.8 μCi/μmol; Perkin Elmer, Boston, MA, USA) was used as substrate, and 100 mM Tris–HCl plus 0–1 mM CaCl2 (pH 7.5) as assay buffer. The reaction mixture contained 20 μl of substrate and either 0.7 pmol of crotoxin subunit B (purified from the venom of Crotalus d. terrificus), or 1 pmol of purified
sPLA2 activity of crotoxin subunit B or purified human synovial sPLA2
The activities of crotoxin subunit B and purified human synovial sPLA2 at various calcium concentrations are shown in Table 1. The two forms of sPLA2 were found to have significant enzymatic activities, even at calcium concentrations comparable to that found intracellularly (0.1 μM).
Externally applied sPLA2 enhances neurotransmitter release in a calcium dependent manner
Crotoxin subunit B (20 ng/ml) was used as the source of Group IIA sPLA2 in these experiments except those shown in Fig. 2B, D, where purified human synovial sPLA2 (final concentration 50 ng/ml) was used. The
Discussion
Secretory phospholipase A2 is associated with synaptosomes and the synaptic vesicle fraction, and it has been suggested that PLA2s may play important roles in synaptic transmission (Nishikawa et al., 1989). The possibility that sPLA2 may affect neurotransmitter release was examined in cultured PC12 cells and hippocampal neurons in the present study. Crotoxin subunit B and purified human synovial sPLA2 were used, together with amperometric and membrane capacitance measurements, to study
Acknowledgements
This work was supported by a grant from the National University of Singapore (R181000054213).
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