Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

作者：Liu, Bin; Cao, Wanxin; Li, Jiping^*; Liu, Jun^*

来源：Cell Calcium, 2018, 76: 62-71.

DOI：10.1016/j.ceca.2018.09.006

• Adenosine triphosphate
• P2Y(2)R-PLC-IP3 pathway
• Lysosomal exocytosis
• Calcium

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague-Dawley rats were established. P2Y(2) receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immuno-labelled in marginal cells of the stria vascular. We found that 30 mu M ATP and 30 mu M uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca2+ concentration ([Ca2+](i),) in the absence of extracellular Ca2+, whereas the response was suppressed by 100 mu M suramin, 10 mu M 1-(6-(17 beta-3-methoxyester-1,3,5(10)-trien-17-yeamino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 mu M 2-aminoethoxydiphenyl borate (2-APB) and 5 mu M thapsigargin (TG), thus indicating that ATP coupled with the P2Y(2)R-PLC-IP3 pathway to evoke Ca2+ release from the endoplasmic reticulum (ER). Incubation with 200 mu M Gly-Phe-beta-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca2+](i) M; this effect was partly inhibited by P2Y(2) R-PLC-IP3 pathway antagonists. After pre-treatment with 5 mu M TG, [Ca2+](i) was significantly lower than that after treatment with P2Y(2)R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca2+ triggers Ca2+ release from ER Ca2+ stores. Baseline [Ca2+]i, declined after treatment with the Ca2+ chelator 50 mu M bis-(aminophenolxy) ethane-N,N,N',N'-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 mu M ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 mu M vacuolin-1. Release of ATP and beta-hexosaminidase both increased after treatment with 200 mu M GPN and 5 mu M TG, but decreased after incubation with 50 mu M BAPTA-AM, 4 IU/ml apyrase and 5 mu M vacuolin-1. We suggest that ATP triggers Ca2+ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca2+ triggers Ca2+ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca2+ signals indirectly via the P2Y(2)R-PLC-IP3 pathway.

• 出版日期2018-12
• 单位上海交通大学