Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells.
The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+.
Lurtz
Monica M
MM
Dept. of Biology, Georgia State Univ., MSC 8L0389, 33 Gilmer St SE Unit 8, Atlanta, GA 30303-3088, USA. biomll@langate.gsu.edu
Louis
Charles F
CF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D007476:Ionophores / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010539:Permeability / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D012756:Sheep
D002147:Calmodulin; D018031:Connexin 43; D004791:Enzyme Inhibitors; D007476:Ionophores; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium