Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation1

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 ± 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 ± 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E2 and 6-keto-PGF1α (+250 ± 85% and +259 ± 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE2 and 6-keto-PGF1α. The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE2 or 6-keto-PGF1α levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE2 and/or PGI2 as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca2+ influx, increase Ca2+/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

The Biosynthesis of Salicylic Acid in Potato Plants1

Spraying potato (Solanum tuberosum L.) leaves with arachidonic acid (AA) at 1500 μg mL−1 led to a rapid local synthesis of salicylic acid (SA) and accumulation of a SA conjugate, which was shown to be 2-O-β-glucopyranosylsalicylic acid. Radiolabeling studies with untreated leaves showed that SA was synthesized from phenylalanine and that both cinnamic and benzoic acid were intermediates in the biosynthesis pathway. Using radiolabeled phenylalanine as a precursor, the specific activity of SA was found to be lower when leaves were treated with AA than in control leaves. Similar results were obtained when leaves were fed with the labeled putative intermediates cinnamic acid and benzoic acid. Application of 2-aminoindan-2-phosphonic acid at 40 μm, an inhibitor of phenylalanine ammonia-lyase, prior to treatment with AA inhibited the local accumulation of SA. When the putative intermediates were applied to leaves in the presence of 2-aminoindan-2-phosphonic acid, about 40% of the expected accumulation of free SA was recovered, but the amount of the conjugate remained constant.

Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis.

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.

A 12R-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression

A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can account for the biosynthesis. Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12R-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12S-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10R-3H] and [10S-3H]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydrogen from the 10-carbon of arachidonate. This result is compatible with 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a P450-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share ≤50% identity to other human lipoxygenases. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.

Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1α from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2–4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-l-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase—programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.