No abstract

We investigated, in monocytic leukemia U937 cells, the effects of docosahexaenoic acid (DHA; 22:6 n-3) on calcium signaling and determined the implication of phospholipase C (PLC) and protein kinase C (PKC) in this pathway. DHA induced dose-dependent increases in [Ca 2ϩ ] i , which were contributed by intracellular pool, via the production of inositol-1,4,5-triphosphate (IP 3 ) and storeoperated Ca 2ϩ (SOC) influx, via opening of Ca 2ϩ release-activated Ca 2ϩ (CRAC) channels. Chemical inhibition of PLC, PKC␥, and PKC␦, but not of PKC␤ ⌱/II, PKC␣, or PKC␤I, significantly diminished DHA-induced increases in [Ca 2ϩ ] i . In vitro PKC assays revealed that DHA induced a ϳ2-fold increase in PKC␥ and -␦ activities, which were temporally correlated with the DHA-induced increases in [Ca 2ϩ ] i . In cell-free assays, DHA, but not other structural analogs of fatty acids, activated these PKC isoforms. Competition experiments revealed that DHA-induced activation of both the PKCs was dose-dependently inhibited by phosphatidylserine (PS). Furthermore, DHA induced apoptosis via reactive oxygen species (ROS) production, followed by caspase-3 activation. Chemical inhibition of PKC␥/␦ and of SOC/CRAC channels significantly attenuated both DHA-stimulated ROS production and caspase-3 activity. Our study suggests that DHA-induced activation of PLC/IP 3 pathway and activation of PKC␥/␦, via its action on PS binding site, may be involved in apoptosis in U937 cells.Epidemiological, clinical, and experimental studies have established that ingestion of n-3 polyunsaturated fatty acids (n-3 PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), present in marine fish oils, exert beneficial effects in several autoimmune and inflammatory disorders (Calder, 2004) Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.039792. ABBREVIATIONS:PUFA, polyunsaturated fatty acid; AA, arachidonic acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; CRAC, Ca 2ϩ release-activated Ca 2ϩ channels; DAG, diacylglycerol; DHA, docosahexaenoic acid; DHA-meth, DHA methyl ester; DHE, dihydroethidine; Dic8, 1,2-dioctanoyl-sn-glycerol; DPEA, cis-7,10,13,16,19-docosapentaenoic acid; DTT, dithiothreitol; EPA, eicosapentaenoic acid; GF 109203X, bisindolylmaleimide I; Gö -6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; HBDDE, 2,2Ј,3,3Ј,4,4Ј-hexahydroxy-1,1Ј-biphenyl-6,6Ј-dimethanol dimethyl ether; IP 3 , inositol-1,4,5-trisphosphate; LSD, least-significant difference; MBP, myelin basic protein; MMP, mitochondrial membrane potential; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidyl-L-serine; ROS, reactive oxygen species; [1][2][3]propoxy]-4-methoxyphenethyl)-1H-imidazole; SOC, store-operated Ca 2ϩ channels; TA9, tyrphostin A9; TG, thapsigargin; U-73122, 1-[6-[[17␤-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]...