Arachidonic acid-induced dye uncoupling in rat cortical astrocytes is mediated by arachidonic acid byproducts

Abstract

Arachidonic acid (AA) induced a concentration- and time-dependent reduction in gap junction-mediated dye coupling between cultured astrocytes. The effect was greatly diminished by inhibition of cyclooxygenases and lipoxygenases. The action of a low concentration of AA (5 μM) was also prevented by Ca 2+-free extracellular solution or a high concentration of melatonin, a potent free radical scavenger, but not by Nω-nitro- l-arginine, a nitric oxide (NO) synthase inhibitor. Thus, this effect may depend on Ca 2+ influx and oxygen free radicals but not on NO generation. Cellular uncoupling induced by a high (100 μM), but not a low, AA concentration was rapidly reversed by washing with albumin containing solution. After reversal from 5 min but not 2.5 min inhibition with a high AA concentration dye coupling between astrocytes became refractory to a low concentration of AA, suggesting desensitization of the response elicited by a low concentration of the fatty acid. Dye uncoupling occurred without changes in levels and state of phosphorylation (immunoblotting and 32 P -incorporation) of connexin43, the main astrocyte gap junctional protein. However, maximal cell uncoupling induced by a low (Slow action) but not by a high (Fast action) AA concentration was paralleled by a reduction in connexin43 (immunofluorescence) at cell-to-cell contacts. It is proposed that the AA-induced dye uncoupling is mediated by byproducts that induce rapid channel closure or slow removal of connexin43 gap junctions.