Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt,and p65 signals induced by sPLA2 in inflammatory mouse paw edema model

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A₂ (PLA₂). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA₂s. Among these, we found EUK-8 is a potent inhibitor with its IC₅₀ value ranges 0.7–2.0 µM for sPLA₂s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA₂. In vivo effect of EUK-8 was studied in HSF-sPLA₂-induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA₂ activity, it also plays an important role in the regulation of sPLA₂-induced cell signaling cascades. Apart from the sPLA₂ inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.     

Chronic lithium administration potentiates brain arachidonic acid signaling at rest and during cholinergic activation in awake rats

Studies were performed to determine if the reported 'proconvulsant' action of lithium in rats given cholinergic drugs is related to receptor-initiated phospholipase A2 signaling via arachidonic acid. Regional brain incorporation coefficients k* of intravenously injected [1-14C]arachidonic acid, which represent this signaling, were measured by quantitative autoradiography in unanesthetized rats at baseline and following administration of subconvulsant doses of the cholinergic muscarinic agonist, arecoline. In rats fed LiCl for 6 weeks to produce a therapeutically relevant brain lithium concentration, the mean baseline values of k* in brain auditory and visual areas were significantly greater than in rats fed control diet. Arecoline at doses of 2 and 5 mg/kg intraperitoneally increased k* in widespread brain areas in rats fed the control diet as well as the LiCl diet. However, the arecoline-induced increments often were significantly greater in the LiCl-fed than in the control diet-fed rats. Lithium's elevation of baseline k* in auditory and visual regions may correspond to its ability in humans to increase auditory and visual evoked responses. Additionally, its augmentation of the k* responses to arecoline may underlie its reported 'proconvulsant' action with cholinergic drugs, as arachidonic acid and its eicosanoid metabolites can increase neuronal excitability and seizure propagation.     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids

Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca²⁺ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference.     

Integrative role of cPLA with COX-2 and the effect of non-steriodal anti-inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.     

PDMS微柱阵列型拓扑结构基底增强HepG2细胞TRPV1、TRPV4通道表达及功能响应性

制备了微柱名义直径为4μm或10μm,名义间距为4μm或7μm,名义高度为4μm的聚二甲基硅氧烷微柱阵列型拓扑结构基底,研究了HepG2细胞与拓扑结构基底复合后细胞瞬时受体电位通道TRPV1、TRPV4在基因和蛋白水平的表达及其功能响应性。细胞TRPV1和TRPV4在基因水平表达的评价采用定量PCR技术进行;TRPV1和TRPV4在蛋白水平的表达以免疫印迹和免疫荧光染色确认;TRPV1和TRPV4功能响应性的研究系以TRPV1和TRPV4激动剂辣椒素和4α-佛波醇-12,13-二葵酸酯刺激细胞,采用钙离子染料钙绿-1结合激光共聚焦显微技术记录钙内流动态过程,以钙内流荧光响应幅度及阳性响应比率进行评价。实验结果表明,在四种拓扑结构基底上细胞TRPV1和TRPV4的mRNA表达量均显著高于平面基底上相应值。免疫印迹实验证实了TRPV1和TRPV4在蛋白水平的表达,且拓扑结构基底上TRPV1和TRPV4免疫荧光染色强度较之平面基底相应值明显增高或趋于增高。在激动剂作用下,TRPV1介导的钙内流表现为快速去敏感化(25秒内)的瞬态内流,且拓扑结构基底上阳性响应细胞比例或相对荧光响应幅度较之平面基底相应值增高;而拓扑结构基底上细胞TRPV4阳性响应细胞比例和相对荧光响应幅度较之平面基底均全面明显升高。上述结果表明,TRPV介导的离子信号可能是基底拓扑结构优化HepG2细胞功能表型的重要信号机制。     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Participation of preoptic area TRPV4 ion channel in regulation of body temperature

Transient receptor potential vanilloid 4 (TRPV4) ion channel is a non-selective cation channel and its role in cutaneous thermosensation is emerging. It is expressed in many areas of the brain including the preoptic area (POA)/anterior hypothalamus which is the key neural site for thermoregulation. The present study was conducted to find out the role of TRPV4 ion channel in the POA in thermoregulation. Rats preimplanted with guide cannulae with indwelling styli 2.0 mm above the POA received TRPV4 agonist/antagonist/isotonic saline injections bilaterally in the POA using an injector cannula in three separate groups of six rats each. Body temperature (Tb) was recorded telemetrically by preimplanted radio transmitter in the peritoneal cavity. The injection of TRPV4 agonist (GSK1016790A) in the POA decreased Tb while its antagonist (RN1734) increased Tb. Immunohistochemical localization showed presence of TRPV4 ion channel in the POA. The results of the present study suggest that TRPV4 ion channels in the POA may play an important role in thermoregulation.     

Involvement of multiple protein kinases in cPLA2 phosphorylation, arachidonic acid release, and cell death in in vivo and in vitro models of 1-methyl-4-phenylpyridinium-induced parkinsonism – the possible key role of PKG

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP⁺-induced cytosolic phospholipase A₂ (cPLA₂) activation of dopaminergic neurons. MPP⁺ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca²⁺]_i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA₂, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF₃), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA₂ on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA₂ phosphorylation at Ser505. Inhibitors of cPLA₂ and PKG increased viability and reduced MPP⁺-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA₂ phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA₂.     

热环境对大鼠纹状体神经元细胞膜的损伤作用

采用原代培养的大鼠纹状体神经元,施予43℃热环境处理1h。用气,质联用的方法测定细胞膜和细胞中的脂肪酸水平,主要是花生四烯酸的水平;荧光偏振法测细胞膜流动性,用[^3H]花生四烯酸大肠杆菌膜检测细胞内磷脂酶A2活性。发现细胞内大量存在的脂肪酸水平在热环境处理前后没有明显差别,而花生四烯酸的水平明显升高。热处理造成细胞膜的流动性明显降低,同时明显增加了神经元内磷脂酶A2的活性。表明热处理明显影响神经元细胞膜的流动性和磷脂代谢,进而影响细胞膜的功能,而热对细胞膜的损伤作用可能就是热致神经元损伤的重要事件。     

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with ³H-arachidonic acid, ionomycin and Ca²⁺-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC₅₀ = 1.6 ± 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca²⁺-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 μM), a blocker of PLA₂, completely inhibited bradykinin-induced arachidonic acid release. The B₂ bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B₁-selective agonist DesArg⁹-bradykinin was inactive and the B₁-selective antagonist [Leu⁸]DesArg⁹-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 μM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 μM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA₂ by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca²⁺-calmodulin appears to be the prominent activator of PLA₂ in this system. © 1996 Wiley-Liss, Inc.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.     

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.     

Rapid activity-dependent sprouting of optic fibers into a local area denervated by application of beta-bungarotoxin in goldfish tectum

The retinotectal projection is known to be capable of extensive long-term expansion of connections, but it is not known how fast such changes can occur or what triggers sprouting of terminals. We studied sprouting of optic fibers into an area denervated by local microinjection of beta-bungarotoxin (β-BTX), a specific presynaptic neurotoxin with phospholipase A₂ activity that destroys nerve terminals at the neuromuscular junction. After injection of 0.1 pmol of β-BTX, the optic terminals fired spontaneously with decreasing amplitude and became silent within 1 to 2 h. Outside the injection zone, the retinotectal map was normal, so the silent zone was associated with a scotoma in the visual field. Horseradish peroxidase (HRP) staining of the entire optic nerve showed a denervated region at the injection site with beaded, degenerating fibers at its edge. Between 3 and 9 days later, optic units were recorded within the injection zone whose receptive fields lay just outside the scotoma in the visual field, indicating that intact surrounding terminals had sprouted into the area. These sprouts made functional connections, as indicated by field potential recordings and current source-density analysis. At this time, HRP staining also demonstrated retinal innervation within the injection zone. By 12 days, normal maps with no scotoma were recorded and HRP staining was normal at the injection site, indicating that the β-BTX-damaged fibers had regenerated to reclaim their tectal sites. The results show that the retinotectal projection of goldfish is very dynamic, since intact optic fibers can sprout into adjacent vacant postsynaptic territory within 2 to 3 days, much faster than previously reported. In a final experiment, we showed that this sprouting is activity-dependent, since it could be prevented by blocking retinal activity with intraocular tetrodotoxin (TTX) during the first 2 days postinjection, even though TTX block of activity does not block regeneration in this system. One possible mechanism for this rapidly triggered sprouting is that arachidonic acid liberated by β-BTX acts as a sprouting factor to attract surrounding healthy fibers into the denervated region but requires activity at the terminals to be effective. © 1996 John Wiley & Sons, Inc.     

Role of iPLA(2) in the regulation of Src trafficking and microglia chemotaxis

Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca(2+) -independent phospholipase A(2) (iPLA(2)) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA(2) by 4-bromoenol lactone (BEL) or iPLA(2) knockdown exerted a significant inhibition on phosphatidylinositol-3-kinase (PI3K) activation and chemotaxis. Further examination revealed that iPLA(2) knockdown abrogated Src activation, which is required for PI3K activation and chemotaxis. Colocalization studies showed that cSrc-GFP was retained in the endosomal recycling compartment (ERC) in iPLA(2) knockdown cells, but the addition of arachidonic acid (AA) could restore cSrc trafficking to the plasma membrane by allowing the formation/release of recycling endosomes associated with cSrc-GFP. Using BODIPY-AA, we showed that AA is selectively enriched in recycling endosomes. These results suggest that AA is required for the cSrc trafficking to the plasma membrane by controlling the formation/release of recycling endosomes from the ERC.