Arachidonic acid-induced dye uncoupling in rat cortical astrocytes is mediated by arachidonic acid byproducts.
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 microM) was also prevented by Ca2+-free extracellular solution or a high concentration of melatonin, a potent free radical scavenger, but not by Nomega-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor. Thus, this effect may depend on Ca2+ influx and oxygen free radicals but not on NO generation. Cellular uncoupling induced by a high (100 microM), 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 32P-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.
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Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.jcslab@genes.bio.puc.cl
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D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D001253:Astrocytes / Q000187:drug effects*; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects*; D005456:Fluorescent Dyes; D005609:Free Radicals; D007546:Isoquinolines; D008084:Lipoxygenase / Q000378:metabolism; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012710:Serum Albumin, Bovine
D005456:Fluorescent Dyes; D005609:Free Radicals; D007546:Isoquinolines; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D016718:Arachidonic Acid; C017475:lucifer yellow; D008084:Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D002118:Calcium