Purchase this article with an account.
M. Shahidullah, N. A. Delamere; Studies on Connexin Hemichannels in the Porcine Nonpigmented Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1451.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Gap junctions between the pigmented (PE) and nonpigmented ciliary epithelium (NPE) act as communication pathways between the two cells. Consistent with this notion, we detect connexin 43 (Cx 43) at the apical surface of the porcine NPE where the cells contact the PE. We also detect another connexin, Cx 50, in the NPE. However, Cx 50 occurs at the NPE basolateral surface, where it faces the aqueous humor and is apparently unpaired. In other tissues, unpaired connexons are able to form active hemichannels that connect the cytoplasm with the extracellular space. Here we probed for possible hemichannel function.
Porcine NPE was established in primary culture. Connexins were detected by immunolocalization and immunoblot. To probe for hemichannel activity, cells were grown on permeable inserts and incubated with the DNA intercalating agent propidium iodide (PI) (Mol. wt. 668.4) or fluorescein dextran (FDex) (Mol. wt 4000), both of which are membrane impermeable. PI or FDex was detected by fluorescence microscopy and fluorimetry.
Under control conditions, only trace amounts of PI entered cultured NPE. Because hemichannel opening is increased by extracellular calcium removal, the cells were incubated 30 min in calcium-free buffer. This increased PI entry significantly. Importantly, PI uptake was abolished by a gap junction inhibitor 18 alpha glycyrrhetinic acid (100 µM). Consistent with the known sensitivity of hemichannels to extracellular pH, PI entry to the NPE was significantly higher at pH 7.6 than at pH 7.4 or lower. FDex did not enter the cultured NPE either in control or calcium-free medium, indicating size-limited solute entry.
The findings are consistent with the ability of NPE cells to form functional connexin hemichannels. Since Cx 50 is localized to the unapposed basolateral surface of the NPE, we suggest it may act as a hemichannel conduit between the cytoplasm and the aqueous humor.
This PDF is available to Subscribers Only