Reduced 5-lipoxygenase metabolism of arachidonic acid in macrophages rrom 1,25-dihydroxyvitamin D3-deficient rats.

The peripheral blood monocyte (PBM) migrates into tissues and differentiates into mature tissue macrophages. Previous investigations from our laboratory have demonstrated that PBM have reduced 5-lipoxygenase (5-LO) metabolism of arachidonic acid (AA) and 5-LO activating protein (FLAP) expression as compared to differentiated alveolar macrophages (AM). Moreover, PBM differentiated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) displayed increased leukotriene synthesis and a parallel increase in FLAP expression.

Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.

Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the translocation of the enzyme from a soluble to a pelletable fraction in thoroughly disrupted granulocytic cells. While immunoelectron microscopy has identified the nuclear membrane as the site at which 5-LO, as well as 5-LO activating protein (FLAP), are localized in activated cells, the locale of soluble 5-LO in unstimulated cells could not be established by this technique. We asked whether the nucleus might also be the site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cells to address this question.

Increases in 5-lipoxygenase activating protein expression account for enhanced capacity for 5-lipoxygenase metabolism that accompanies differentiation of peripheral blood monocytes into alveolar macrophages.

The capacity for 5-lipoxygenase (5-LO) metabolism of endogenous arachidonic acid (AA) is greater in alveolar macrophages (AM) than in their circulating precursors, peripheral blood monocytes (PBM); however, the ability of PBM to metabolize exogenous AA to 5-LO products is comparable to that of AM. In the present study, we examined the enzymatic mechanisms by which 5-LO metabolism of AA is altered during differentiation of PBM in the lung. Resting human AM exhibited greater steady-state levels of 5-LO (7-fold) and LTA4 hydrolase (2-fold) proteins than autologous PBM; moreover, they differed from PBM in that they contained a significant amount of 5-LO associated with the particulate fraction.

Augmented expression of cytosolic phospholipase A2 during phenotypic transformation of cultured type II pneumocytes.

Over time in culture, rat type II alveolar epithelial cells (AEC) demonstrate increased levels of unesterified arachidonic acid (AA) and increased prostanoid synthesis, while assuming certain morphological and biochemical characteristics of the type I cell phenotype. The objective of this study was to elucidate the enzymatic mechanism(s) responsible for increased AA accumulation in this model. Cells were examined both early in culture (2 days), when they retained type II cell features, and later in culture (7 days), when they are known to express a number of type I cell characteristics.

Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation.

Both the cytosolic phospholipase A2 and 5-lipoxygenase enzymes redistribute from the high-speed supernatant to a particulate fraction upon cell activation with associated leukotriene synthesis, but the subcellular site to which these enzymes translocate is not known. In this study, we disrupted resting and ionophore A23187-stimulated rat peritoneal macrophages by N2 cavitation and separated lysates into nuclear, cytosolic, and crude membrane fractions; these were then examined by immunoblot analysis for their contents of immunoreactive cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase activating protein. 5-Lipoxygenase activating protein was localized predominantly in the nuclear fraction of both resting and activated cells, while both cytosolic phospholipase A2 and 5-lipoxygenase redistributed from the cytosol fraction in resting cells to the nuclear fraction in activated cells.

Transcellular eicosanoid synthesis in cocultures of alveolar epithelial cells and macrophages.

We have examined the importance of the relative capacities for arachidonic acid (AA) liberation and oxygenation as determinants of the magnitude of transcellular eicosanoid synthesis in cocultures of [3H]AA-prelabeled alveolar macrophages (AM) and [14C]AA-prelabeled alveolar epithelial cells (AEC). Taking advantage of our previous observation that over time in culture, AEC exhibit increases in AA release (plateau at day 4) that precede increases in cyclooxygenase capacity (maximal at day 7), we studied cocultures of freshly harvested AM together with AEC at culture days 2, 4, and 7. In this model, ionophore stimulation resulted in transcellular eicosanoid synthesis via the bidirectional transfer of free AA, with AM synthesizing their exclusive 5-lipoxygenase products leukotriene B4 and 5-hydroxyeicosatetraenoic from AEC-derived AA, and AEC synthesizing their exclusive cyclooxygenase metabolites prostaglandin E2 and prostacyclin from AM-derived AA.

Phospholipase A2 in alveolar type II epithelial cells: biochemical and immunologic characterization.

The enzyme phospholipase A2 (PLA2) catalyzes phospholipid hydrolysis and is thought to play important roles in surfactant synthesis and in the generation of lipid mediators, including eicosanoids and platelet-activating factor. This study sought to characterize PLA2 in rat type II pneumocytes by biochemical and immunologic means. Type II cells were found to contain an alkaline-active, Ca(2+)-dependent PLA2 activity predominantly localized to cytosol fraction.

Hydrogen peroxide increases the availability of arachidonic acid for oxidative metabolism by inhibiting acylation into phospholipids in the alveolar macrophage.

Reactive oxygen species stimulate metabolism of arachidonic acid (AA) to eicosanoids in a variety of cells and tissues, yet the pathway(s) by which oxidants increase the availability of AA for oxidative metabolism are not known. Thus, we explored the effects of hydrogen peroxide (H2O2) on deacylation and reacylation of AA to determine the enzymatic mechanism(s) by which this oxidant increases levels of free, unesterified AA, and thereby its oxidative metabolism to eicosanoids, in the rat alveolar macrophage (AM). Over the range from 0.

Decreased airway mucosal prostaglandin E2 production during airway obstruction in an animal model of asthma.

Heaves is a respiratory disorder of horses and ponies characterized by bouts of acute airway obstruction and airway hyperresponsiveness. We measured prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) production in vitro in tracheal epithelium obtained from six affected horses at the time of acute airway obstruction as compared with six matched control horses. Strips of epithelium and subepithelial tissue were prepared and stimulated with A23187, histamine, and bradykinin.

Protein kinase C activation modulates arachidonic acid metabolism in cultured alveolar epithelial cells.

Cultured alveolar type II cells can liberate esterified arachidonic acid (AA) and metabolize it predominantly via the cyclooxygenase pathway, and their capacity to do so increases as they alter their phenotype over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation.

Membrane association of active 5-lipoxygenase in resting cells. Evidence for novel regulation of the enzyme in the rat alveolar macrophage.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in granulocytic cells have shown that activation of 5-LO involves its Ca(2+)-dependent translocation from cytosol to membrane compartments. However, little information exists about the molecular regulation of 5-LO in macrophages, even though these cells comprise the resident effector cell population of most organs.

Epithelial strips: an alternative technique for examining arachidonate metabolism in equine tracheal epithelium.

We have developed an alternative method for examining equine tracheal epithelial arachidonic acid (AA) metabolism that utilizes strips of pseudostratified columnar epithelium attached to a layer of elastic tissue 80 to 130 microns thick. We compared the responses of this preparation with those of enzymatically dispersed suspensions of tracheal epithelium obtained from the same animal. Strips incubated with [3H]AA incorporated 40.

Basal activation of protein kinase C in rat alveolar macrophages: implications for arachidonate metabolism.

Alveolar macrophages (AM) exhibit numerous functional differences from other mononuclear phagocyte populations, even though they are derived from a common circulating monocytic precursor. Yet no differences in fundamental signaling mechanisms uniquely expressed by AM have been elucidated to date. Protein kinase C (PKC) is one signal transduction mechanism thought to have an important role in regulating macrophage function and about which little information exists for AM.

Differential dependence on protein kinase C of arachidonic acid metabolism stimulated by hydrogen peroxide and by zymosan in the alveolar macrophage.

The dependence on protein kinase C (PKC) of arachidonic acid (AA) metabolism stimulated by the biologically important oxidant H2O2, as compared to zymosan particles, was investigated in the rat alveolar macrophage. The PKC inhibitor staurosporine markedly reduced AA release and eicosanoid synthesis stimulated by zymosan, but only slightly inhibited AA release and metabolism induced by H2O2. Furthermore, in macrophages depleted of PKC by extended exposure to phorbol 12-myristate 13-acetate, AA release in response to zymosan was greatly inhibited, whereas that stimulated by H2O2 was attenuated to a significantly lesser degree.

Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro.

Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation.

Glucocorticoids fail to inhibit arachidonic acid metabolism stimulated by hydrogen peroxide in the alveolar macrophage.

We have previously demonstrated that the biologically important oxidant hydrogen peroxide (H2O2) triggers release and metabolism of arachidonic acid (AA) in the alveolar macrophage (AM). In this study, we evaluated the ability of glucocorticoids to inhibit rat AM AA metabolism stimulated by H2O2, as compared to the particulate zymosan. Methylprednisolone and other glucocorticoids failed to significantly inhibit release of AA stimulated by H2O2, while markedly reducing AA release in response to zymosan.

Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung.

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC).

Complex effects of in vitro hyperoxia on alveolar macrophage arachidonic acid metabolism.

Metabolites of arachidonic acid (AA) released into bronchoalveolar lavage fluid of animals exposed to hyperoxia have previously been implicated as mediators of pulmonary oxygen toxicity. The alveolar macrophage (AM) represents an important potential source of these eicosanoids. We have therefore investigated the effects of in vitro hyperoxia (95% O2/5% CO2) versus normoxia (95% air/5% CO2) on the metabolism of AA in the AM of the rat.

Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung.

The present study was undertaken to investigate the changes in arachidonic acid (AA) metabolism which accompany rat macrophage (m phi) differentiation in the lung in order to determine whether these changes occur in the alveolar space or in the pulmonary interstitium, as well as the mechanisms responsible for such changes. Metabolism of endogenous and exogenous AA by cultured m phi obtained from the peritoneum (PM), the pulmonary interstitium (IM), and the alveolar spaces (AM) was examined by using HPLC and RIA. Although PM and AM released similar amounts of endogenous AA in response to both ionophore A23187 and the particulate zymosan, PM metabolized AA predominantly to cyclooxygenase (CO) products, whereas AM produced predominantly 5-lypoxygenase (5-LO) metabolites.

Multiple defects in arachidonate metabolism in alveolar macrophages from young asymptomatic smokers.

This study was undertaken to localize and determine the relative importance of potential biochemical defects in the release and metabolism of arachidonic acid (AA) in alveolar macrophages (AMs) from asymptomatic smokers. Using high-performance liquid chromatography and radioimmunoassay, we compared the metabolism of both endogenously released and exogenously supplied AA in AMs and autologous peripheral blood monocytes (PBMs) from nine healthy nonsmokers and eight healthy smokers. AMs from both groups incorporated similar amounts of radiolabeled AA into cellular lipids.

Inhibition of rat lung glutathione synthesis attenuates hypoxic pulmonary vasoconstriction and the associated leukotriene C4 production.

Lipoxygenase metabolites of arachidonic acid have been proposed as possible mediators of hypoxic pulmonary vasoconstriction (HPV) in the rat. Since reduced glutathione (GSH) is a required substrate for the synthesis of the sulfidopeptide eicosanoid leukotriene C4 (LTC4), we reasoned that this specific GSH dependence of LTC4 synthesis might allow us to distinguish between the roles of sulfidopeptide leukotrienes and other 5-lipoxygenase metabolites of arachidonic acid. In the present study we have examined the effect of in vivo pretreatment with the GSH synthesis inhibitor buthionine sulfoximine (BSO) on both the hypoxic pressor response and lung leukotriene synthesis in the rat.

Inhibition of alveolar macrophage 5-lipoxygenase metabolism by auranofin.

We have examined the effect of the oral gold compound auranofin (AF) on calcium ionophore A23187-induced arachidonic acid metabolism in the rat alveolar macrophage. Both reverse-phase high performance liquid chromatographic and radioimmunoassay analyses revealed that AF dose-dependently inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid synthesis in a parallel fashion with an IC50 approximately 4.3 micrograms/ml.

Different patterns of arachidonate metabolism in autologous human blood monocytes and alveolar macrophages.

As peripheral blood monocytes (PBM) differentiate into tissue macrophages, they undergo a variety of functional changes. One such difference which has been described is an enhanced metabolism of arachidonic acid (AA) via the 5-lipoxygenase (5-LO) pathway in alveolar macrophages (AM) as compared to PBM. In order to elucidate a possible mechanism for this difference, we compared the metabolism of endogenously released AA mobilized by agonists and of exogenously supplied fatty acid in adherent autologous PBM and AM obtained from six normal subjects.