PMID- 12091436
OWN - NLM
STAT- MEDLINE
DCOM- 20020715
LR  - 20151119
IS  - 0146-0404 (Print)
IS  - 0146-0404 (Linking)
VI  - 43
IP  - 7
DP  - 2002 Jul
TI  - Lysosomal Ca(2+) stores in bovine corneal endothelium.
PG  - 2341-50
AB  - 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.
FAU - Srinivas, Sangly P
AU  - Srinivas SP
AD  - School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 
      47405, USA. srinavas@indiana.edu
FAU - Ong, Angeline
AU  - Ong A
FAU - Goon, Leanne
AU  - Goon L
FAU - Goon, Levina
AU  - Goon L
FAU - Bonanno, Joseph A
AU  - Bonanno JA
LA  - eng
GR  - EY 11107/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Invest Ophthalmol Vis Sci
JT  - Investigative ophthalmology & visual science
JID - 7703701
RN  - 0 (Dipeptides)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (fura-PE3)
RN  - 21438-66-4 (glycylphenylalanine 2-naphthylamide)
RN  - 85166-31-0 (Inositol 1,4,5-Trisphosphate)
RN  - F30N4O6XVV (Acridine Orange)
RN  - SY7Q814VUP (Calcium)
RN  - TSN3DL106G (Fura-2)
RN  - UT0S826Z60 (Uridine Triphosphate)
SB  - IM
MH  - Acridine Orange
MH  - Animals
MH  - Calcium/*metabolism
MH  - Cattle
MH  - Cells, Cultured
MH  - Dipeptides/pharmacology
MH  - Endothelium, Corneal/drug effects/*metabolism
MH  - Fluorescent Dyes
MH  - Fura-2/*analogs & derivatives
MH  - Hydrogen-Ion Concentration
MH  - Inositol 1,4,5-Trisphosphate/metabolism
MH  - Lysosomes/drug effects/*metabolism
MH  - Uridine Triphosphate/pharmacology
EDAT- 2002/07/02 10:00
MHDA- 2002/07/16 10:01
CRDT- 2002/07/02 10:00
PHST- 2002/07/02 10:00 [pubmed]
PHST- 2002/07/16 10:01 [medline]
PHST- 2002/07/02 10:00 [entrez]
PST - ppublish
SO  - Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2341-50.