Supplementary Material for: Kainate Receptors Mediate Regulated Exocytosis of Secretory Phospholipase A2 in SH-SY5Y Neuroblastoma Cells

Secretory phospholipase A2 (sPLA2) isoforms are widely expressed in the brain and spinal cord. Group IIA sPLA2 (sPLA2-IIA) has been shown to stimulate exocytosis and release of neurotransmitters in neuroendocrine PC12 cells and neurons, suggesting a role of the enzyme in neuronal signaling and synaptic transmission. However, the mechanisms by which sPLA2 is itself released, and a possible relation between glutamate receptors and sPLA2 exocytosis, are unknown. This study was carried out to elucidate the effects of glutamate receptor agonists on exocytosis of sPLA2-IIA in transfected SH-SY5Y neuroblastoma cells. sPLA2-IIA enzyme was packaged in fusion-competent vesicles and released constitutively or upon stimulation, suggesting regulated secretion. The signal peptide of sPLA2-IIA is required for its vesicular localization and exocytosis. External application of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) induced vesicular exocytosis and release of sPLA2-IIA. UBP 302, a GluR5-specific KA receptor antagonist, abolished the effect of KA, confirming the role of KA receptors in mediating sPLA2-IIA secretion. Moreover, KA-induced sPLA2-IIA secretion is dependent on Ca2+ and protein kinase C. Together, these findings provide evidence of a link between glutamate receptors and regulated sPLA2 secretion in neurons that may play an important role in synaptic plasticity, pain transmission and neurodegenerative diseases.

分泌型磷脂酶A2（secretory phospholipase A2, sPLA2）同工酶在大脑与脊髓中广泛表达。IIA型sPLA2（sPLA2-IIA）已被证实可在神经内分泌PC12细胞与神经元中诱导胞吐作用并促进神经递质释放，提示该酶在神经元信号传导与突触传递中发挥功能。然而，sPLA2自身的释放机制，以及谷氨酸受体与sPLA2胞吐之间的潜在关联，目前仍不明确。本研究旨在阐明谷氨酸受体激动剂对转染后的SH-SY5Y神经母细胞瘤细胞中sPLA2-IIA胞吐过程的影响。研究发现，sPLA2-IIA酶被包裹于具有融合能力的囊泡中，可通过组成型分泌或受刺激后释放，提示其存在调控型分泌途径。sPLA2-IIA的信号肽对其囊泡定位与胞吐过程不可或缺。外源施加α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA）与红藻氨酸（kainate, KA）可诱导囊泡胞吐并释放sPLA2-IIA。GluR5特异性红藻氨酸受体拮抗剂UBP 302可阻断KA的上述效应，证实红藻氨酸受体在介导sPLA2-IIA分泌中的作用。此外，KA诱导的sPLA2-IIA分泌依赖于钙离子与蛋白激酶C。综上，本研究结果提供了谷氨酸受体与神经元内调控型sPLA2分泌之间存在关联的证据，该关联可能在突触可塑性、疼痛传导与神经退行性疾病中发挥重要作用。