TY - JOUR
T1 - The influence of thapsigargin on na,k-ATPase activity in cultured nonpigmented ciliary epithelial cells
AU - Mito, Takezo
AU - Kuwahara, Soichiro
AU - Delamere, Nicholas A.
N1 - Funding Information:
This work was supported by USPHS Research Grant No. EY06915, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc. We wish to thank Dr Miguel Coca-Prados for his generous gift of the ODM2 cell line used in this study.
PY - 1995
Y1 - 1995
N2 - Experiments were conducted to test the influence of thapsigargin on the Na,K-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 μM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.
AB - Experiments were conducted to test the influence of thapsigargin on the Na,K-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 μM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.
KW - ATP hydrolysis
KW - Human ciliary epithelium
KW - Na,K-ATPase
KW - Thapsigargin
UR - https://www.scopus.com/pages/publications/0029114046
UR - https://www.scopus.com/pages/publications/0029114046#tab=citedBy
U2 - 10.3109/02713689508998492
DO - 10.3109/02713689508998492
M3 - Article
C2 - 8529400
AN - SCOPUS:0029114046
SN - 0271-3683
VL - 14
SP - 651
EP - 657
JO - Current Eye Research
JF - Current Eye Research
IS - 8
ER -