Elsevier

Neuroscience

Volume 121, Issue 4, 7 November 2003, Pages 891-898
Neuroscience

Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

https://doi.org/10.1016/S0306-4522(03)00525-6Get rights and content

Abstract

Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

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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