Inability of skin enzyme preparations to biosynthesize arachidonic acid from linoleic acid

The lack of any information as to the origin of epidermal arachidonic acid, an important precursor of eicosanoids in the epidermis, prompted us to determine in vitro whether or not microsomal preparations from rat and guinea pig epidermis possess the delta 6 and delta 5 desaturase activities. The incubations were performed in parallel with microsomal preparations from liver of these animals where activities for these enzymes have previously been reported. The conversions of radioactive fatty acids were determined after methylation and separation of the 14C-fatty acid methyl esters by argentation thin layer chromatography. Data from these studies demonstrated that delta 5 desaturase activity is markedly lower in guinea pig liver than in rat liver. Interestingly, preparations from rat and guinea pig epidermis at all concentrations tested lacked the capacity to transform either linoleic acid into gammalinolenic acid or dihomogammalinolenic acid into arachidonic acid. This observation implies that arachidonic acid that is present in the epidermal phospholipids is biosynthesized elsewhere endogenously and transported to the epidermis for esterification into the phospholipids. The site of this biosynthesis is presumably the liver and the mode of transport to the epidermis remains to be determined. These studies indicate arachidonic acid per se as an essential fatty acid for the epidermis.     

Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes

The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo synthesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate (14C-LA) into 18:3(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) (AA) was noted. The conversion of non-radiolabeled 18:3(n-6) or 20:2(n-6) was also examined and the pattern of metabolites synthesized suggests that the preferred metabolic pathway for conversion of linoleic acid into arachidonic acid is via the classically described pathway in which a delta 6 desaturase constitutes the initial reaction. Although cultured skin fibroblasts are known to convert linoleic acid into arachidonic acid, the current study demonstrates that cultured epidermal keratinocytes can also avidly metabolize exogenous linoleic acid. The ability of cultured keratinocytes, and not of whole epidermis in vivo, to convert linoleic acid into arachidonic acid suggests that specific enzymatic activities may be induced by the tissue culture system itself. Hence, findings of metabolic capabilities in cultured cells may not necessarily be extrapolated to the in vivo situation.     

Transfomration of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by mouse peritoneal macrophages.

Mouse peritoneal macrophages were incubated at 37 degrees C for 30 min with arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE₂ and PGI₂ (as 6-keto-PGF_1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI₂, PGE₂ and LTC₄ were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

gamma-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters

Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.     

Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx.

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2+ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Ca2+ was added to the incubation medium. When arachidonic acid release was examined in Ca(2+)-depleted cells, the response took place only in presence of external Ca2+. Verapamil, a voltage-dependent Ca2+ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2+ influx is functionally linked to arachidonic acid release and hence to phospholipase A2 activation in mouse peritoneal macrophages.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.     

The effect of ethanol on arachidonic acid metabolism in the murine peritoneal macrophage

Exposure to ethanol in man has been linked to an alteration of the immune surveillance system and reduced ability of the macrophage to undergo phagocytosis. Since ethanol has been suggested to alter membrane function and inhibit the production of calcium ionophore stimulated synthesis of prostaglandins and leukotrienes by the human neutrophil and transformed murine mast cell, the dose response effect of ethanol on the biosynthesis of icosanoids by the peritoneal macrophage during zymosan phagocytosis was studied. Peritoneal macrophages from two inbred strains of mice derived from a common stock (HS) and selected for sensitivity to ethanol (short sleep [SS]/long sleep [LS]) were studied. Zymosan phagocytosis was found to lead to synthesis of LTC4 (70 ng/10(6) cells), 6-keto-PGF1 alpha (5 ng/10(6) cells) and PGE2 (3 ng/10(6) cells). For the HS macrophage, ethanol caused a dose dependent inhibition of these lipid mediators as well as inhibition of phagocytosis and release of beta-hexosaminidase. However, a difference was observed in arachidonate metabolism stimulated by phagocytosis between the LS and SS mice below 100 mM ethanol. The SS mouse had a 50% inhibition of cyclooxygenase products at 86 mM ethanol with no inhibition of lipoxygenase metabolites. The LS mice had a trend suggesting increased lipoxygenase metabolites below 100 mM ethanol. At these levels of ethanol which can be found in man, these results suggest there may be differential production of lipid mediators under genetic control.     

Involvement of external calcium in the release of arachidonic acid by mouse peritoneal macrophages

The present investigation was undertaken to study the potential role of extracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages. Both in phorbol ester-treated and in Ca2(+)-depleted cells, a rapid release of arachidonic acid was seen in direct response to added Ca2+. The response was directly dependent on the extracellular Ca2+ concentration, with a Ca2+ threshold of 100 nM. These results support the notion that arachidonic acid release in macrophages is functionally coupled to influx of external calcium.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.     

Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.     

Ethanol induces release of arachidonic acid but not synthesis of eicosanoids in mouse peritoneal macrophages

Exposure of mouse peritoneal macrophages to ethanol induces a rapid release of arachidonic acid to the extracellular medium. All major classes of phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contribute to this release. Ethanol-induced mobilization of arachidonic acid occurs by deacylation, but it is not accompanied by eicosanoid synthesis. These data suggest that at least two signals are necessary for the release and metabolism of arachidonic acid. Ethanol also activates a phospholipase C which hydrolyzes only phosphatidylinositol, and not its phosphorylated derivatives.     

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4.

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.     

Production of arachidonic acid and linoleic acid metabolites by human bronchoalveolar lavage cells.

Fatty acid-derived inflammatory mediators are considered to play an important role in airway hyperresponsiveness of asthmatic patients. The pulmonary macrophage may be an important source for these mediators in airway tissue. We investigated the metabolism of arachidonic acid and linoleic acid by human bronchoalveolar lavage cells, mainly comprising pulmonary macrophages. Arachidonic was mainly metabolized by 5-lipoxygenase, giving rise to the formation of leukotriene B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). Linoleic acid was converted to 5 major metabolites, including the 9-hydroxy and 13-hydroxy derivatives, 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE). The formation of HODEs could be inhibited by cyclooxygenase inhibitors as well as lipoxygenase inhibitors, indicating that both enzymic species play a role in the generation of HODEs.     

Conversion of linoleic acid and arachidonic acid by skin epidermal lipoxygenases

Two different lipoxygenases have been identified in human and rat epidermis. One lipoxygenase has a (n-9)-specificity, converts arachidonic acid into 12-hydroxyeicosatetraenoic acid (12-HETE), and has been described by several investigators. Linoleic acid is not a substrate for this enzyme. The other lipoxygenase, with (n-6)-specificity, converts arachidonic acid into 15-HETE and linoleic acid into 13-hydroxyoctadecadienoic acid (13-HOD). Especially the latter lipoxygenase is thought to be involved in the regulation of the differentiation of the skin cells into a proper water-barrier layer. Linoleate is supposed to be the physiological substrate; this fatty acid is especially present in characteristic sphingolipids with unique structures.     

Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.