Merck

Calcium entry via connexin hemichannels in lens epithelium.

Experimental eye research (2015-01-20)
Amritlal Mandal, Mohammad Shahidullah, Nicholas A Delamere
RESUMEN

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.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Sodium pyrophosphate tetrabasic, ≥95%
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
Sodium bicarbonate, BioXtra, 99.5-100.5%
Sigma-Aldrich
Sodium bicarbonate, Hybri-Max, powder, suitable for hybridoma, ≥99.5%
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, for molecular biology, ≥97.0%
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, ≥97.0%
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioXtra, ≥97 .0%
Supelco
Calcium standard for AAS, analytical standard, 1.000 g/L Ca+2 in hydrochloric acid, traceable to BAM
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioUltra, for molecular biology, ≥99.0% (T)
Sigma-Aldrich
Sodium bicarbonate, tested according to Ph. Eur.
Sigma-Aldrich
Glycerol solution, 83.5-89.5% (T)
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Propidium iodide, ≥94% (HPLC)
Sigma-Aldrich
HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
HEPES, BioXtra, suitable for mouse embryo cell culture, ≥99.5% (titration)
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Sodium orthovanadate, 99.98% trace metals basis
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Sodium orthovanadate, ≥90% (titration)
Supelco
Glycerin, Pharmaceutical Secondary Standard; Certified Reference Material
SAFC
HEPES
SAFC
HEPES
USP
Sodium bicarbonate, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Sodium bicarbonate, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99.7%
Sigma-Aldrich
Glycerol solution, puriss. p.a., 86-89% (T)
Sigma-Aldrich
Sodium hydrogencarbonate, −40-+140 mesh, ≥95%
Sigma-Aldrich
Sodium bicarbonate, ReagentPlus®, ≥99.5%, powder