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

Abstract 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 immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the absence of extracellular Ca 2+ , whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y 2 R-PLC-IP3 pathway to evoke Ca 2+ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca 2+ ] I ; this effect was partly inhibited by P2Y 2 R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca 2+ ] i was significantly lower than that after treatment with P2Y 2 R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca 2+ triggers Ca 2+ release from ER Ca 2+ stores. Baseline [Ca 2+ ] i declined after treatment with the Ca 2+ chelator 50 μM bis-(aminophenolxy) ethane- N , N , N ʹ, N ʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM 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 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca 2+ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca 2+ triggers Ca 2+ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca 2+ signals indirectly via the P2Y 2 R-PLC-IP3 pathway.