title: Lysosomal Ca(2+) stores in bovine corneal endothelium.
abstract: OBJECTIVE
  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.
authors.affiliation: School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA. srinavas@indiana.edu
authors.forename: Sangly P
authors.initials: SP
authors.lastname: Srinivas
authors.forename: Angeline
authors.initials: A
authors.lastname: Ong
authors.forename: Leanne
authors.initials: L
authors.lastname: Goon
authors.forename: Levina
authors.initials: L
authors.lastname: Goon
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
mesh_terms: D000165:Acridine Orange; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D004151:Dipeptides; D005456:Fluorescent Dyes; C098106:fura-PE3; C041865:glycylphenylalanine 2-naphthylamide; D015544:Inositol 1,4,5-Trisphosphate; D000165:Acridine Orange; D002118:Calcium; D016257:Fura-2; D014544:Uridine Triphosphate