Abstract

P 074

Calcium, a modulator of proliferation: the role of blocking voltage gated calcium channels (Cav) in human lens epithelial cells (hLEC)

Meißner A., Noack T.
Institut für Physiologie, Universität Rostock

Objective: Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage gated calcium channels. T-type und L-type calcium channels are substantially present in hLEC and total calcium currents are inhibited by Mibefradil. Here, the hypothesis was tested if calcium influx via Cav channels regulates proliferation in epithelial cells.
Methods: Cell proliferation was determined by cell culture assays, using the L- and T-type Cav channel blockers Mibefradil and Verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench-technique. Western blot experiments were accomplished under standard conditions, using antibodies against c-Raf, MAPK 3 and CaMKII. PCR experiments were performed after the isolation of RNA or genomic DNA from HLE-B3 cells after treatment with different concentrations of Mibefradil or Verapamil.
Results: Mibefradil as well as Verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of c-Raf, MAPK 3 and CaMKII was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. Under control conditions the addition of Manganese quenched the fluorescence signal rapidly: after 1 min fluorescence was reduced to 57±3% (n=3; p<0.05). However, in the presence of 10-5 M Mibefradil the signal decreased only to 67±2% (n=3; p<0.05) after 1 min. Verapamil also impaired the calcium influx, but less than Mibefradil.
Conclusions: We conclude that the transmembrane influx of Ca²⁺ through Cav channels contributes to the regulation of hLEC proliferation, identifying Cav channel blockers as potential therapeutic substances in ocular diseases.
Supported by DFG No 296/5

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