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Sangly P. Srinivas, Angeline Ong, Leanne Goon, Levina Goon, Joseph A. Bonanno; Lysosomal Ca2+ Stores in Bovine Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2341-2350.
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purpose. Acidic organelles, including Golgi bodies and lysosomes, are known to operate as Ca2+ storage sites in many cell types. This study demonstrates the presence of Ca2+ stores in lysosomes of bovine corneal endothelial cells (BCECs) and examines their interaction with Ins(1,4,5)P3-sensitive Ca2+ stores.
methods. Glycyl-l-phenylalanine-β-naphthylamide (GPN) was used to release Ca2+ from lysosomes by inducing their selective osmotic swelling. Ca2+ 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 μM) in cultured BCECs produced an increase in free cytosolic Ca2+ ([Ca2+]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 [Ca2+]i transients similar to those in cultured BCECs; however, the peak [Ca2+]i increase was smaller (∼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 ([Ca2+]o) at 0 and 1.2 mM (n = 10). Neither pretreatment with thapsigargin (5 μM) nor with U73122 (a phospholipase C inhibitor; 10 μM) blocked the peak GPN response (n = 6). Exposure to 20 μM monensin produced a [Ca2+]i increase with [Ca2+]o at 0 and 1.2 mM and also reduced the subsequent peak response to GPN (n = 6).
conclusions. GPN-sensitive lysosomal Ca2+ stores, distinct from Ins(1,4,5)P3-sensitive Ca2+ 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)P3. The GPN stores also induce [Ca2+]o influx in response to their depletion.
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