Monica M Lurtz1, Charles F Louis. 1. Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521, USA. monica.lurtz@ucr.edu
Abstract
PURPOSE: To determine whether the purinergic receptor-mediated, delayed transient inhibition of lens cell-to-cell communication is due to the protein kinase C (PKC)-catalyzed phosphorylation of connexin (Cx)43. METHODS: The functional activity of gap junctions was determined in the presence of various pharmacologic agents by injecting fluorescent dye into a single cell in a confluent monolayer of HeLa cells that had been stably transfected with either wild-type or mutant Cx43, and the number of cells taking up dye was determined. RESULTS: Application of adenosine triphosphate (ATP) to Cx43-transfected HeLa cells resulted in a delayed, transient decrease in cell-to-cell transfer of fluorescent dye similar to the authors' previous report in sheep lens epithelial cell cultures. The ATP-mediated, delayed, transient decrease in dye transfer was prevented by the inhibition of PKC or phospholipase C, but not by calmodulin inhibition or by preloading the cells with BAPTA (bis-(o-aminophenoxy)-N,N'N'-tetraacetic acid). This functional inhibition of Cx43 cell-to-cell dye transfer was sustained in the presence of the nonhydrolyzable ATP analogue AMP-PNP (adenyl-5'-yl imidophosphate), the ectonucleotidase inhibitor ARL 67156, or the protein phosphatase inhibitor okadaic acid. In experiments in HeLa cells transfected with Cx43(Delta 257), a Cx43 C terminus truncation mutant, or Cx43(S368A), a Cx43 point mutant, cell-to-cell coupling was unaffected by the addition of ATP. CONCLUSIONS: The results indicate the essential role of serine 368 in the ATP-dependent inhibition of Cx43. This novel mechanism of regulating Cx43 most likely plays an important role in maintaining the microcirculation that is essential for the movement of water and solutes in the intact lens.
PURPOSE: To determine whether the purinergic receptor-mediated, delayed transient inhibition of lens cell-to-cell communication is due to the protein kinase C (PKC)-catalyzed phosphorylation of connexin (Cx)43. METHODS: The functional activity of gap junctions was determined in the presence of various pharmacologic agents by injecting fluorescent dye into a single cell in a confluent monolayer of HeLa cells that had been stably transfected with either wild-type or mutant Cx43, and the number of cells taking up dye was determined. RESULTS: Application of adenosine triphosphate (ATP) to Cx43-transfected HeLa cells resulted in a delayed, transient decrease in cell-to-cell transfer of fluorescent dye similar to the authors' previous report in sheep lens epithelial cell cultures. The ATP-mediated, delayed, transient decrease in dye transfer was prevented by the inhibition of PKC or phospholipase C, but not by calmodulin inhibition or by preloading the cells with BAPTA (bis-(o-aminophenoxy)-N,N'N'-tetraacetic acid). This functional inhibition of Cx43 cell-to-cell dye transfer was sustained in the presence of the nonhydrolyzable ATP analogue AMP-PNP (adenyl-5'-yl imidophosphate), the ectonucleotidase inhibitor ARL 67156, or the protein phosphatase inhibitor okadaic acid. In experiments in HeLa cells transfected with Cx43(Delta 257), a Cx43 C terminus truncation mutant, or Cx43(S368A), a Cx43 point mutant, cell-to-cell coupling was unaffected by the addition of ATP. CONCLUSIONS: The results indicate the essential role of serine 368 in the ATP-dependent inhibition of Cx43. This novel mechanism of regulating Cx43 most likely plays an important role in maintaining the microcirculation that is essential for the movement of water and solutes in the intact lens.