Evidence for Connexin Hemichannel Activity in Porcine Ocular Nonpigmented Ciliary Epithelium

Abstract

ObjectiveRecent studies have shown that unpaired connexons form active hemichannels connecting the cytoplasm to the extracellular space. In porcine ciliary body we detected connexin 50 (Cx 50) at the aqueous humor (AH)‐facing basolateral surface and connexin 43 at the apical surface of nonpigmented ciliary epithelium (NPE).MethodsConnexins were detected by immunological study. To probe for hemichannel activity, cells were cultured 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.ResultsUnder control conditions, only traces of PI entered cultured NPE. PI entry was markedly increased in cells incubated with calcium‐free buffer. FDex did not enter the cultured NPE either in control or calcium‐free medium, indicating size‐limited entry. PI entry also was dependent on [pH]o; significantly higher at pH 7.6 than at pH 7.4 or lower. PI uptake was inhibited by a gap‐junction‐specific blocker 18[alpha] glycyrrhetinic acid (100 [mu]M).ConclusionsThe findings are consistent with the ability of NPE cells to form functional 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 AH. EY006915