Micromolar levels of intracellular calcium reduce gap junctional permeability in lens cultures

Abstract

Purpose: To investigate in bovine and embryonic chicken lens cultures the effects of elevated intracellular calcium on the permeability of gap junctions. To determine the changes in intracellular calcium using fura-2. To detect any changes in the phosphorylation of connexin43 after ionophore treatment.

Methods: Lucifer yellow was micro-injected into individual cells, and dye spread to neighboring cells was evaluated. Intracellular calcium levels were measured using the calcium indicator, fura-2. Cultures were also labeled with 32P-orthophosphate followed by immunoprecipitation with antibodies specific for the gap junction protein, connexin43.

Results: Bovine lens cultures incubated in the presence of either A23187 or ionomycin showed a reduction in intercellular dye transfer. The intracellular calcium concentrations in bovine cells were increased from a mean value of 0.11 +/- .009 microM in the controls to a mean of 0.40 +/- .073 microM with ionomycin treatment. Subsequent addition of EGTA to the medium decreased the intracellular calcium concentrations to a mean of 0.26 +/- .113 microM and reversed the inhibition of dye spread found with ionomycin. With ionomycin in the medium, the phosphorylated form of connexin43 was not as prominent as in the controls. In contrast, these same treatments had no detectable effect on junctional permeability in the embryonic chicken lens cultures. Dye spread was equally extensive and rapid under control and ionophore conditions, even though fura studies showed an elevation in intracellular calcium levels.

Conclusions: In the bovine cultures, physiologically relevant changes in the levels of cytoplasmic calcium markedly reduced dye transfer. The increase in cytoplasmic calcium was correlated with a change in the phosphorylation level of connexin43. The regulation of junctional communication in the chick lens cultures appears to differ significantly from that in the bovine system.