Research reportArachidonic acid-induced dye uncoupling in rat cortical astrocytes is mediated by arachidonic acid byproducts
Introduction
Cortical astrocytes couple to each other, both in vivo and in vitro, by numerous gap junctions which confer them a functional syncytial organization 22, 34, 37. Gap junction channels permit the intercellular exchange of current-carrying ions (e.g., K+), second-messenger molecules (e.g., cyclic nucleotides, Ca2+, inositol 1,4,5-trisphosphate) and various metabolites 4, 9. Consequently, astrocyte gap junctions are believed to create a buffering system that is responsible for the dissipation of focal extracellular K+ and neurotransmitter concentration increases resulting from intense neural activity 39, 48, 49. Therefore, gap junctional communication between astrocytes facilitates the normal functioning of neurons and protects them from excitotoxicity of neurotransmitters. In agreement, it has been recently reported that astrocytic gap junctional communication decreases neuronal vulnerability to a cell insult [7]. Moreover, gap junctions mediate the propagation of calcium waves between astrocytes after receptor 12, 13, 29, 70or mechanic stimulation [11], providing an extraneuronal cell-to-cell signaling pathway.
Gap junction channels span the plasma membrane of two adjacent cells. The contribution of each cell is termed an hemichannel or connexon, each of which is constituted of six protein subunits called connexins. At least 14 rodent connexins have been cloned from various tissues and each one is named according to its molecular mass predicted from the cloned DNA sequence (e.g., the 43–45 kDa protein with a predicted molecular mass of 43-kDa found in heart gap junctions is called connexin43, Cx43) [6]. Studies in primary cultures of brain astrocytes 17, 27have shown that Cx43 is the major gap junction channel protein in astrocytes.
The activation of astrocytic receptors by endogenous ligands, including norepinephrine [28], glutamic acid [21], and endothelins [68], affects the degree of coupling between adjoining cells. Consequently, the functional state of gap junction channels can be modulated by cyclic AMP, Ca2+ and diacylglycerol-dependent pathways 4, 9; the receptor-mediated alterations may use these second messengers. Moreover, two diffusible neurotransmitters, nitric oxide (NO) 8, 54and arachidonic acid (AA) 28, 69and its derivative anandamide [69], have been shown to reduce gap junctional communication between astrocytes. Nonetheless, their mechanism of action remains largely unknown.
The AA is a fatty acid that directly affects the functional state of various molecules, including the protein kinase C (PKC) [42], calcium channels [45]and (Na+/K+)-ATPase [64]. Moreover, various biologically active products are yielded through its metabolism via a cyclooxygenase-dependent pathway forming prostaglandins and thromboxanes, and a lipoxygenase-dependent pathway generating leukotrienes, lipoxins, and related compounds [60]. Several of these compounds affect the functional state of a variety of membrane proteins, including plasma membrane Ca2+ and K+ channels 38, 51, 58, 60and the Na+/Ca2+ exchanger [50], as well as second messenger pathways 14, 15, 30, 35. Moreover, metabolites of AA, such as prostaglandin D2 increase the activity of NO synthase [71], suggesting a link between AA metabolism and NO synthesis. Other AA byproducts generated by cyclooxygenase and lipoxygenase-dependent pathways are oxygen free radicals which are highly reactive compounds that affect a variety of cell functions [61].
Gap junctions between hepatocytes 52, 55, cells of lacrimal glands [26], neonatal cardiac myocytes 23, 43, 67, a rat mammary tumor cell line [74], Novikoff cells [41], and retinal horizontal cells [44]are also blocked by AA. While gap junction inhibition in lacrimal cells has been proposed to occur by direct interaction of AA on the channels [26], in others systems the effect seems to be mediated, at least in part, by byproducts of the AA metabolism 23, 43, 52, 55. It is unknown whether astrocyte uncoupling is induced directly by AA or indirectly by its metabolites.
The present work was undertaken to elucidate possible mechanisms that could explain the AA-induced reduction in dye coupling in primary cultures of rat cortical astrocytes. It was found that AA induced a profound reduction in gap junctional communication between astrocytes in a manner independent of NO generation, but dependent on AA metabolism through cyclooxygenases and lipoxygenases. The AA-induced astrocyte uncoupling was not correlated with changes in levels or state of phosphorylation of Cx43. Both high and low concentrations of AA induced similar maximal effects but with different time courses and through different mechanisms.
Section snippets
Reagents
Arachidonic acid, melatonin, Nω-nitro-l-arginine, indomethacin, nordehydroguaiaretic acid (NDGA), 1,3-dimethyl-2-thiourea (DMTU), phorbol 12-myristate 13 acetate (PMA), phenylmethylsulfonyl fluoride (PMSF), dimethyl sulfoxide, and Lucifer yellow were purchased from Sigma (St. Louis, MO, USA). Luzindole was from Alexis (San Diego, CA, USA). F-12 medium, minimal essential medium (MEM), fetal bovine serum, horse serum, penicillin, trypsin-EDTA, bovine pancreas DNAase I and streptomycin were from
AA induces a dose and time-dependent reduction in dye coupling
In confluent cultures, astrocytes were coupled to an average of 15±3 cells (Fig. 1A) and the incidence of dye coupling was 100%. In 6 out of 12 experiments, the initial dye uncoupling response after the addition of 5 μM AA was quite heterogeneous; a few minutes (5 min) after the addition of the fatty acid, 15–25% of the cells were completely uncoupled, while the others still showed a normal degree of communication with neighboring cells. In the other six experiments, the reduction in dye
Discussion
In the present work, we studied the possible mechanism of action of AA-induced inhibition of astrocyte gap junctional communication. We found that uncoupling of astrocytes was mediated by products generated in AA cyclooxygenase and lipoxygenase metabolic pathways, which predominantly produced either a rapid closure of gap junction channels or a slow cellular redistribution of the main channel forming protein, Cx43.
Using the dye coupling technique, we found that a high AA concentration (100 μM)
Acknowledgements
The authors thank Dr. M.V.L. Bennett for his suggestions. This work was partially supported by Fondecyt grants 2960001 (to A.D.M.) and 1960559 (to J.C.S.). The data in this paper are from a thesis to be submitted in partial fulfillment of the requirements for the Degree of Doctor in Sciences (A.D. Martı́nez) in the Pontificia Universidad Católica de Chile.
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