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Evidence for differential activation of arachidonic acid metabolism in formylpeptide- and macrophage-activation-factor-stimulated guinea-pig macrophages.

ABSTRACT: Alterations of phospholipid and arachidonic acid metabolism were studied by treatment of guinea-pig peritoneal-exudate macrophages with chemotactic peptide, formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) and macrophage activation factor (MAF). The chemotactic peptide caused a rapid rearrangement in inositol phospholipids, including a breakdown of polyphosphoinositides within 30s, followed by a resultant formation of phosphatidylinositol (PI), diacylglycerol, phosphatidic acid and non-esterified arachidonic acid within 5 min. In addition to these sequential alterations, arachidonic acid was released mainly from PI. On the other hand, MAF induced a slow liberation of arachidonic acid, mainly from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) by phospholipase A2 after the incubation period of 30 min, but not any rapid changes in phospholipids. Treatment of macrophages for 15 min with fMet-Leu-Phe produced the leukotrienes (LTs) B4, C4 and D4, prostaglandins (PG) E2 and F2 alpha and thromboxane (TX) B2. In contrast, MAF could not stimulate the production of arachidonic acid metabolites during the incubation period of 15 min, but could enhance that of PGE2, PGF2 alpha, TXB2 and hydroxyeicosatetraenoic acids at 6 h. However, the stimulated formation of LTs was not detected at any time. These results indicate that the effects of fMet-Leu-Phe on both phospholipid and arachidonic acid metabolism are very different from those mediated by MAF.

SUBMITTER: Homma Y

PROVIDER: S-EPMC1145106 | biostudies-other | 1985 Aug

REPOSITORIES: biostudies-other

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Project description:The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-beta-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca(2+) ionophore A23187. The effect of calmodulin antagonists on the incorporation of [(32)P]P(i) or [(3)H]glycerol into glycerolipids as well as on the redistribution of [(14)C]glycerol or [(3)H]arachidonic acid in [(14)C]glycerol- or [(3)H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [(32)P]P(i) incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [(32)P]P(i) into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [(32)P]P(i) incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [(3)H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [(3)H]glycerol into PtdA and PtdIns was greatly enhanced. But [(3)H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [(3)H]glycerol was maximally activated by 10mum-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [(14)C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [(14)C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [(3)H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [(3)H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-(3)H]diacylglycerol and non-esterified [(3)H]-arachidonic acid was also enhanced, but the increase in [(3)H]arachidonic acid was only observed at concentrations between 1 and 50mum. [Arachidonate-(3)H]PtdIns was not significantly affected. The activated formation of [arachidonate-(3)H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.

Project description:Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < -0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), "lipid metabolism" was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.

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Evidence for differential activation of arachidonic acid metabolism in formylpeptide- and macrophage-activation-factor-stimulated guinea-pig macrophages.

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