Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats
Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats
复制标题
ATP 的溶酶体胞吐作用与新生大鼠纹状体血管边缘细胞中的 P2Y(2) 受体偶联
DOI：
10.1016/j.ceca.2018.09.006
复制
发表时间：
2018-12-01
期刊：
影响因子：
4
通讯作者：
Liu, Jun
中科院分区：
文献类型：
--
作者：
Liu, Bin;Cao, Wanxin;Liu, Jun
三磷酸腺苷（ATP）在新生大鼠血管纹边缘细胞中以溶酶体囊泡形式存在，但ATP释放的机制尚不清楚。建立1日龄Sprague-Dawley大鼠边缘细胞原代培养物。在血管纹边缘细胞中免疫标记了P_2Y（2）受体和1，4，5-三磷酸肌醇（IP_3）受体。我们发现，30 μ M ATP和30 μ M尿苷三磷酸（UTP）引起细胞内Ca 2+浓度的显著增加（[Ca ~（2+）]（i）），而100 μ M苏拉明、10 μ M 1-（6-（17 β-3-甲氧基酯-1，3，5（10）-三烯-17-基氨基）-己基）-1H-吡咯-2，5-二酮（U-73122），100 μ M 2-氨基乙氧基二苯基硼酸盐（2-APB）和5 μ M毒胡萝卜素（TG），这表明ATP与P2 Y（2）R-PLC-IP 3途径偶联，引起内质网（ER）的Ca 2+释放。与200 μ M Gly-Phe-beta-naphthylamide（GPN）孵育选择性破坏溶酶体，并引起[Ca ~（2+）]（i）M显著增加;这种作用可被P_2 Y（2）R-PLC-IP_3通路拮抗剂部分抑制。用5 μ M TG预处理后，[Ca ~（2+）]（i）显著低于在相同条件下用P_2 Y（2）R-PLC-IP_3通路拮抗剂处理后的[Ca ~（2+）]（i），从而表明溶酶体Ca ~（2+）触发ER Ca ~（2+）库的Ca ~（2+）释放。用Ca 2+螯合剂50 μ M双-（氨基苯氧基）乙烷-N，N，N '，N'-四乙酸乙酰氧基甲酯（BAPTA-AM）和4 IU/ml腺苷三磷酸双磷酸酶治疗后，基线[Ca 2 +]i下降。30 μ M ATP通过溶酶体胞吐减少奎纳克林阳性囊泡的数量，而溶酶体的数量没有变化。然而，溶酶体胞吐作用显着抑制预处理5 μ M vacuolin-1。用200 μ M GPN和5 μ M TG处理后，ATP和β-氨基己糖苷酶的释放均增加，但用50 μ M BAPTA-AM、4 IU/ml腺苷三磷酸双磷酸酶和5 μ M vacuolin-1孵育后，ATP和β-氨基己糖苷酶的释放减少。我们认为，ATP触发Ca 2+从ER释放，从而有助于通过溶酶体胞吐分泌溶酶体ATP。溶酶体储存的Ca 2+通过IP 3受体直接触发Ca 2+从ER释放，而溶酶体ATP通过P2 Y（2）R-PLC-IP 3途径间接引发Ca 2+信号。
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.