Calcium entry via connexin hemichannels in lens epithelium

Abstract

Exposure to hyposmotic solution causes release of ATP from lens cells via hemichannels. Because hemichannel opening feasibly could swamp the cells with calcium, we carried out studies to measure the magnitude of the increase in cytoplasmic calcium concentration caused by hemichannel opening. In studies on porcine lens epithelial cells in primary culture, propidium iodide (PI) uptake was measured as an index of hemichannel opening. PI uptake was increased significantly in cells exposed to hyposmotic solution. The PI increase under hyposmotic conditions was suppressed by GAP 27, a connexin inhibitor peptide. In studies on cells loaded with Fura-2, continuous exposure to hyposmotic solution caused a cytoplasmic calcium concentration increase that peaked within ∼30 s then remained elevated at or below the peak response for more than 60 min. The peak calcium concentration was 186 ± 2.3 nM compared to a baseline value of 98.0 ± 1.4 nM. The calcium concentration increased a lot further in cells exposed to A23187 (2.5 μM) or the sodium–calcium exchange inhibitor SN-6 (10 μM) added after the onset of the calcium rise in hyposmotic solution. The cytoplasmic calcium increase in hyposmotic solution was abolished by GAP 27. Calcium returned to baseline in cells exposed to hyposmotic solution then treated with GAP 27 starting 2 min after the onset of the calcium rise. The calcium increase in hyposmotic solution did not occur when calcium was eliminated from the bathing medium. The responses to hyposmotic and hyperosmotic stress were different. There was no detectable increase in calcium or PI entry in cells exposed to hyperosmotic solution (500mOsm). In summary, GAP 27-sensitive accumulation of PI by cultured lens epithelium points to connexin hemichannel opening and associated calcium entry. Even though connexins form channels with a large carrying capacity, calcium entry does not increase the cytoplasmic calcium concentration beyond a tolerable physiological range.