Assessment of connexin hemichannel involvement in air-stimulated ATP release.

Abstract

<p>Uptake of the connexin permeable dye lucifer yellow (LY) was measured to confirm that connexin hemichannels open in response to air exposure and are blocked by the same drugs that prevent ATP release. Cells were pre-incubated with ATP release inhibitors using the same procedure as for the pharmacological analysis assays. LY (500 µM final concentration) was added in the media after air exposure or a change of half the media (control). Sixty minutes later, cultures were rinsed twice with media containing 1-octanol (2 mM) to remove residual LY, but minimize leakage of absorbed LY. LY levels in the cell lysates were then measured. Air exposure significantly increased LY uptake compared to control and the same drugs that inhibited air-stimulated ATP release prevented this increase in LY uptake. This indicates that connexin hemichannels are open during air-stimulated ATP release. Values represent 8 or 9 wells per group +/− SEM, * <i>p</i><0.05 vs. control.</p