Lysosomal Ca(2+) stores in bovine corneal endothelium.
Lysosomal Ca(2+) stores in bovine corneal endothelium.
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发表时间：
2002-07
影响因子：
4.4
通讯作者：
S. P. Srinivas;A. Ong;Leanne Goon;Levina Goon;J. Bonanno
中科院分区：
文献类型：
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作者：
S. P. Srinivas;A. Ong;Leanne Goon;Levina Goon;J. Bonanno
目的酸性细胞器，包括高尔基体和溶酶体，在许多细胞类型中被称为Ca（2+）储存位点。这项研究表明，牛角膜内皮细胞（BCEC）溶酶体中存在Ca（2+），并检查了它们与INS（1,4,5）P（1,4,5）P（3）P（3）敏感Ca（2+）商店的相互作用。方法通过诱导其选择性的渗透肿胀，使用方法从溶酶体释放Ca（2+）从糖基-L-苯丙氨酸甲基丙氨酸（GPN）中释放Ca（2+）。用Fura-2或Fura-Pe3荧光染料测量释放到细胞质中的Ca（2+）。 Acridine Orange（AO）的荧光选择性地隔离为酸性细胞器，用于响应GPN响应于GPN而建立溶酶体的肿胀。在培养的BCEC中暴露于GPN（100-200 microM）的结果导致自由胞质CA（2+）（[CA（2+）]（i）（i））增加，相当于对尿苷Triphophate（UTP）的峰值响应的大约79％，是P2Y激动剂（N = 19）。新鲜分离的角膜的内皮也会产生[CA（2+）]（i）与培养的BCEC中类似的瞬态；但是，峰[Ca（2+）]（i）增加较小（约占对UTP的峰值响应的43％； n = 13）。在培养的BCEC中，对UTP的反应不受GPN在0和1.2 mm处的细胞外钙（[Ca（2+）]（O））（n = 10）的影响。用Thapsigargin（5 microM）或U73122（磷脂酶C抑制剂; 10 microM）预处理均未阻断GPN峰值响应（n = 6）。在0和1.2 mm处，暴露于20 microm monensin在[CA（2+）]（i）随[CA（2+）]（O）增加的情况下增加，也降低了对GPN的随后峰值响应（n = 6）。结论GPN敏感的溶酶体Ca（2+）商店，不同于INS（1,4,5）P（1,4,5）p（3）敏感的Ca（2+）商店，在培养的细胞和新鲜组织中都发现了。这些商店容易受到跨溶酶体和P2激动剂的pH梯度损失的耗尽。后者是通过独立于磷脂酶C（PLC）激活或ins（1,4,5）p（3）的机制发生的。 GPN存储还会诱导[CA（2+）]（O）响应其耗竭。
PURPOSE Acidic organelles, including Golgi bodies and lysosomes, are known to operate as Ca(2+) storage sites in many cell types. This study demonstrates the presence of Ca(2+) stores in lysosomes of bovine corneal endothelial cells (BCECs) and examines their interaction with Ins(1,4,5)P(3)-sensitive Ca(2+) stores. METHODS Glycyl-L-phenylalanine-beta-naphthylamide (GPN) was used to release Ca(2+) from lysosomes by inducing their selective osmotic swelling. Ca(2+) released into the cytoplasm was measured with fura-2 or fura-PE3 fluorescent dyes. Fluorescence of acridine orange (AO), which selectively sequesters into acidic organelles, was used to establish swelling of lysosomes in response to GPN. RESULTS Exposure to GPN (100-200 microM) in cultured BCECs produced an increase in free cytosolic Ca(2+) ([Ca(2+)](i)) equivalent to approximately 79% of the peak response to uridine triphosphate (UTP), a P2Y agonist (n = 19). The endothelium of the freshly isolated cornea also produced [Ca(2+)](i) transients similar to those in cultured BCECs; however, the peak [Ca(2+)](i) increase was smaller ( approximately 43% of the peak response to UTP; n = 13). In cultured BCECs, the response to UTP was unaffected by pretreatment with GPN with extracellular calcium ([Ca(2+)](o)) at 0 and 1.2 mM (n = 10). Neither pretreatment with thapsigargin (5 microM) nor with U73122 (a phospholipase C inhibitor; 10 microM) blocked the peak GPN response (n = 6). Exposure to 20 microM monensin produced a [Ca(2+)](i) increase with [Ca(2+)](o) at 0 and 1.2 mM and also reduced the subsequent peak response to GPN (n = 6). CONCLUSIONS GPN-sensitive lysosomal Ca(2+) stores, distinct from Ins(1,4,5)P(3)-sensitive Ca(2+) stores, are found in both cultured cells and fresh tissue. These stores are susceptible to depletion by the loss of the pH gradient across lysosomes and P2 agonists. The latter occurs through mechanisms independent of phospholipase C (PLC) activation or Ins(1,4,5)P(3). The GPN stores also induce [Ca(2+)](o) influx in response to their depletion.