Hyposmotic stress triggers ATP release from porcine lens via connexin and pannexin hemichannels

Abstract

AbstractPurpose Purinergic receptors in the lens suggest function can be altered by agonists present in aqueous humor. Some agonists may originate from the lens itself. Here, we examine the ability of osmotic shock to trigger ATP release from the intact lens.Methods Porcine lenses were exposed to hyposmotic (200 mOsm) or hyperosmotic (500mOsm) solution and ATP in the bathing medium was measured by luciferase assay. Because hemichannels are permeable to large solutes, the ability of propidium iodide (PI) (MW 668) to enter the epithelium was examined.Results ATP release into the bathing medium was stimulated when lenses were exposed to hyposmotic solution. Hyperosmotic solution did not detectably increase ATP release. Hyposmotic solution‐induced release of ATP was partially suppressed by the connexin hemichannel inhibitor, 18α glycyrrhetinic acid (AGA) or by probenecid, a pannexin hemichannel blocker, and was abolished by AGA and probenecid added together. Consistent with opening of connexin and/or pannexin hemichannels, lenses exposed to hyposmotic solution displayed a ~4 fold increase in the ability of PI to enter the epithelium. In parallel studies, hyposmotic solution was shown to activate a Src family tyrosine kinase (SFK) and cause an SFK‐dependent increase of Na,K‐ATPase activity in the epithelium.Conclusion Hemichannels contribute to ATP release when the intact lens is subjected to hyposmotic shock. The amount of ATP release appeared sufficient to activate purinergic receptors that cause tyrosine kinase‐dependent stimulation of active Na‐K transport. The responses could perhaps signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.