Biomedical subjects

M Peters-Golden

Publications and source records attributed to M Peters-Golden.

At least 73 records · Page 4Linked to original sources

PubMed1994

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. An increase in AA levels at day 7 persisted despite inhibition of AA reacylation, suggesting that differences in deacylation were responsible for differences in free fatty acid levels. These differences in deacylation were not explained by differing susceptibilities to hydrolysis of radiolabeled endogenous lipids from day 2 and day 7 cells. The phospholipase A2 (PLA2) activities at both days in culture were qualitatively similar and typical of the recently described high-molecular-mass cytosolic PLA2 (cPLA2), but activity in day 7 cytosol was threefold greater than that present in day 2 cytosol. A neutralizing anti-cPLA2 antibody reduced the PLA2 activity in day 7 cytosol to the level found in day 2 cytosol. Immunoblot analysis failed to detect expression of low-molecular-mass PLA2 proteins but confirmed that expression of the 97-kDa cPLA2 was greater in day 7 cytosol than in day 2 cytosol. These results indicate that increased levels of unesterified AA in AEC with phenotype altered during culture are due to augmented steady-state expression of cPLA2 and suggest for the first time that expression of cPLA2 is differentiation dependent.

Acylation↗

PubMed1994

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. Importantly, AM contained 40-fold more 5-lipoxygenase activating protein (FLAP) than did PBM, which correlated well with the relative abilities of intact AM and PBM to metabolize endogenous AA to leukotrienes. The FLAP inhibitor MK-886 was unable to block leukotriene synthesis from exogenous AA in the two cell types, despite its ability to completely inhibit 5-LO metabolism of endogenous AA. These observations indicate that, although FLAP is essential for the synthesis of leukotrienes from endogenous AA, perhaps by presenting AA to 5-LO, it is not required for 5-LO metabolism of exogenous AA. The differing roles of FLAP in 5-LO metabolism of endogenous versus exogenous AA are consistent with the conclusion that it is the markedly greater expression of FLAP, rather than of 5-LO, that is primarily responsible for the increased leukotriene synthesis from endogenous AA that accompanies PBM differentiation into AM.

5-Lipoxygenase-Activating Proteins↗

PubMed1993

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. These data demonstrate for the first time coordinate subcellular localization of the key proteins involved in leukotriene synthesis from endogenous arachidonate.

5-Lipoxygenase-Activating Proteins↗

PubMed1993

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. This activity preferred phospholipids containing arachidonate in the sn-2 position and phosphatidylethanolamine (PE) over phosphatidylcholine (PC); it was active at physiologically relevant Ca2+ concentrations and was resistant to dithiothreitol. These biochemical features were suggestive of a high-molecular-weight form of PLA2, and immunoblot analysis of type II cell extracts indeed detected a 97-kDa protein corresponding to a high-molecular-weight PLA2, but no low-molecular-weight pancreatic-type enzyme. Human A549 cells, which share certain features with type II cells, contained a similar PLA2 activity and immunoreactive protein. By contrast, whole rat lung cytosol contained both a 97-kDa PLA2 and a 14-kDa pancreatic-type PLA2, but its activity was typical of the latter in that it lacked sn-2 specificity for arachidonate and was inhibited by dithiothreitol. These results indicate that rat type II as well as human A549 epithelial cells contain a high-molecular-weight PLA2, for which mounting evidence suggests an important role in physiological and pathophysiological situations.

Animals↗

PubMed1993

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. 14C-labeled 5-lipoxygenase products were synthesized by cocultures in the rank order: day 4 > day 2 > day 7, paralleling the relative amounts of unmetabolized AEC-derived [14C]AA. Transcellular flow of [14C]AA to the AM 5-lipoxygenase pathway was also favored when the AEC cyclooxygenase pathway was inhibited with indomethacin. Inhibition of the AM 5-lipoxygenase pathway with the compound MK 886 similarly resulted in the accumulation of unmetabolized AM-derived [3H]AA and greater synthesis of 3H-labeled prostanoids. We conclude that 1) the quantity of unmetabolized AA made available by one cell is an important determinant of the extent to which the fatty acid is utilized for transcellular eicosanoid synthesis by the second cell; 2) the fate of AA derived from a donor cell is influenced by the expression of AA oxygenation pathways in both the donor cell and the recipient cell; and 3) intercellular exchange of AA favors its shunting from an inhibited oxygenation pathway to an uninhibited one.

Animals↗

PubMed1992

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. We therefore examined the levels of 5-LO activity and immunoreactive protein in cytosol and membrane fractions of resident rat alveolar (AM) and peritoneal macrophages (PM) and compared them with the well studied human neutrophil (polymorphonuclear leukocyte). In the resting state, PM resembled polymorphonuclear leukocyte in that most of their cell-free 5-LO activity, as well as protein content, were localized to the cytosol fraction. By contrast, resting AM contained most of their activity and almost half of their immunoreactive protein in the crude membrane fraction. The inability of the drug MK-886 to reverse this membrane association suggested that the 5-LO-activating protein was not the site of binding in the resting cell; however, this drug completely inhibited leukotriene B4 synthesis in ionophore A23187-stimulated AM, indicating that an interaction between 5-LO and 5-LO-activating protein was nonetheless required for product synthesis upon stimulation. Translocation of cytosolic 5-LO protein could not be convincingly demonstrated in A23187-stimulated AM, suggesting that the pool of 5-LO enzyme responsible for product formation originated in the membrane rather than the cytosol fraction of the resting cell. The AM therefore represents the first mammalian cell in which 5-LO has been recovered from the membrane fraction (a) of a resting cell and (b) in active form. These novel findings extend our understanding of the molecular regulation of 5-LO and may be of importance in designing strategies to limit inflammation in the lung and other sites.

Animals↗

PubMed1992

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. The PGE2 and 15-HETE in media from strips was quantitated by radioimmunoassay. 15-HETE above the limits of accurate detection was found in epithelial strips of only two principal animals and in none of the control horses, and the amount of 15-HETE was not increased when strips were stimulated. Epithelial strips from affected horses tended to produce less PGE2 than did strips from control horses, and there was a significant correlation between epithelial PGE2 production and the time taken for affected animals to develop airway obstruction. Subepithelial tissue strips from control horses produced significantly more PGE2 in response to A23187 and bradykinin than did strips from affected horses. We conclude that equine tracheal epithelium is not a significant source of 15-HETE. Airway mucosal PGE2 production is reduced in horses with heaves, which suggests that a relative decrease in this bronchorelaxant substance may be a factor in the pathogenesis of this model of asthma.

Airway Obstruction↗

PubMed1992

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 0.1 to 0.5 mM, H2O2 caused marked time- and dose-dependent inhibition of incorporation of [3H]AA into macrophage phospholipids, whereas calcium ionophore A23187 and zymosan particles did not cause such inhibition. Within this concentration range, there was an almost exact reciprocal correlation between inhibition of [3H]AA acylation and H2O2-stimulated accumulation of free [3H]AA in prelabeled AM cultures. Thimerosal, which blocks AA reacylation but spares deacylation via phospholipase A2 (PLA2), did not affect accumulation of free [3H]AA in prelabeled cells stimulated with H2O2, while markedly augmenting [3H]AA release in response to A23187 and to zymosan. Despite its ability to block AA acylation almost completely, H2O2 did not directly inhibit arachidonoyl CoA synthetase or arachidonoyl CoA:lysophosphatide acyltransferase, which catalyze AA incorporation into phospholipids. However, H2O2 (0.1 to 0.5 mM) markedly depleted AMs of ATP, required for synthesis of the acylation intermediate arachidonoyl CoA, suggesting that this was the means by which H2O2 inhibited acylation. Notably, H2O2 (0.03 to 3 mM) failed to stimulate macrophage PLA2 activity. We conclude that H2O2, in contrast to A23187 and zymosan, inhibits incorporation of AA into phospholipids, and that this represents the major mechanism by which the oxidant increases the availability of free AA for oxidative metabolism in the AM. This may be an important basis for release of eicosanoids in oxidant-induced inflammation and injury of the lung.

Acylation↗

PubMed1991

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. This study was undertaken to assess the state of activation of PKC in cultured resident rat AM compared with resident rat peritoneal macrophages (PM) and the means by which active PKC regulates arachidonic acid (AA) metabolism in the two cell types. As assessed by a histone phosphorylation assay, resting AM, in contrast to PM, exhibited constitutive activation of PKC as evidenced by localization of a majority of PKC activity to the membrane fraction. Ionophore A23187-stimulated release and metabolism of AA were attenuated by depletion of or inhibition of cellular PKC activity in AM but not in PM. In contrast, A23187-stimulated AA metabolism was augmented by activation of PKC to a greater extent in PM than in AM. Results from both cell types indicated that the 5-lipoxygenase pathway was particularly upregulated by PKC activation. We conclude that activation of PKC occurs uniquely during macrophage residence in the alveolar space and that this property as well as the downregulation of PKC which results have profound consequences for the regulation of at least one important macrophage function, the synthesis of bioactive eicosanoids.

Alkaloids↗

PubMed1990

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. Thus, zymosan-stimulated AA metabolism requires active PKC, whereas H2O2-induced metabolism is largely PKC-independent. This provides direct evidence for the existence of two pathways of agonist-stimulated AA metabolism, which differ in their dependence on PKC, in the alveolar macrophage.

Alkaloids↗

PubMed1990

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). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.

Alkaloids↗

PubMed1990

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. IM synthesized a profile of eicosanoids which more closely resembled that of PM. Studies of the metabolism of exogenously supplied AA demonstrated that AM indeed had less CO activity than did PM. PM, but not AM, CO activity decreased during prolonged culture in air, suggesting the possibility that oxidative inactivation of CO plays a role in the decline in CO capacity which accompanies m phi differentiation in the lung. In contrast, the greater expression of 5-LO metabolism in AM than PM did not reflect mere differences in enzyme capacity, since upon activation of protein kinase C with PMA or oleoylacetylglycerol, ionophore-stimulated PM produced amounts of 5-LO products which were comparable to the amounts produced by AM stimulated with A23187 alone. These results indicate that increases in 5-LO metabolism and decreases in CO metabolism accompany rat m phi differentiation in the lung, that these changes occur largely in the alveolar space, and that the increased 5-LO capacity and decreased CO capacity are independently regulated by different mechanisms.

Animals↗

PubMed1990

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. Similarly, methylprednisolone only weakly inhibited synthesis of thromboxane (Tx)B2 stimulated by H2O2, while inhibiting zymosan-induced eicosanoid synthesis to a marked degree. On the other hand, the phospholipase inhibitor mepacrine strongly inhibited AA release and TxB2 formation stimulated by both H2O2 and zymosan, indicating that H2O2 induced AA metabolism is indeed susceptible to pharmacologic inhibition. The failure of glucocorticoids to inhibit AA metabolism stimulated by H2O2 in the AM may in part explain their inability to ameliorate oxidant-mediated lung inflammation and injury.

Animals↗

PubMed1990

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. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung.

Animals↗

PubMed1990

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. Exposure to 95% O2 for up to 72 h did not impair the viability or affect the protein content of cultured AMs. Hyperoxia for 24 to 72 h increased the accumulation of free AA liberated from endogenous stores in cultures of resting AMs. Despite this increase in free AA, no changes in synthesis of thromboxane B2, prostaglandin (PG) E2, PGF2 alpha, leukotriene (LT) B4, or LTC4 were observed in resting AMs exposed to hyperoxia for up to 72 h. This was not due to degradation of eicosanoids in hyperoxia. However, formation of cyclooxygenase metabolites from exogenously supplied AA was reduced in hyperoxia-incubated AMs, suggesting that hyperoxia inhibited the cyclooxygenase enzyme. In AMs stimulated with calcium ionophore A23187, both AA release and synthesis of cyclooxygenase and lipoxygenase eicosanoids were augmented after incubation in hyperoxia for 24 to 72 h. The increase in A23187-stimulated LTB4 synthesis caused by hyperoxia was inhibited by the antioxidants catalase, superoxide dismutase, and the intracellular cysteine loading agent L-2-oxothiazolidine-4-carboxylic acid, suggesting that the augmentation by hyperoxia of A23187-induced AA metabolism was mediated by reactive oxygen metabolites. Thus, hyperoxia has complex effects on AA metabolism in the AM, which include the ability to augment the release of AA and formation of bioactive eicosanoids. These findings support a possible role for eicosanoid synthesis by the AM in the pathogenesis of oxygen toxicity of the lung.

Animals↗

PubMed1989

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. At the same time, AF augmented A23187-induced arachidonate release and cyclooxygenase metabolism. A possible mechanism for the inhibition of 5-lipoxygenase was suggested by the capacity of AF to dose-dependently deplete ATP (IC50 approximately 5.9 micrograms/ml), a cofactor for 5-lipoxygenase. These data indicate that, at therapeutic concentrations, AF acts in vitro as a selective inhibitor of macrophage 5-lipoxygenase metabolism. This likely represents an important mechanism of action of AF in chronic inflammatory disorders.

Adenosine Triphosphate↗

PubMed1989

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. Exogenous AA was metabolized to larger amounts of both cyclooxygenase (CO) and 5-LO products by PBM as compared with AM. Although the two cell types released similar amounts of endogenous AA in response to ionophore A23187, marked differences in the pattern of its metabolism were observed. In PBM, a large proportion of released AA remained unmetabolized, and that which was metabolized was converted predominantly to CO products. In contrast, arachidonate released by AM was efficiently metabolized, predominantly via the 5-LO pathway. Similar results were obtained when cells were stimulated with the particulate zymosan, with PBM synthesizing mainly CO and AM, mainly 5-LO eicosanoids. In addition, culture of PBM for up to 5 days in an aerobic environment did not alter their response to A23187 stimulation. These results suggest that the lesser 5-LO metabolism by PBM than AM is not explained by lesser phospholipase or 5-LO activities, but rather a compartmentalization of the endogenous AA deacylated by phospholipase and the 5-LO enzyme in the PBM. The acquisition of the capacity to metabolize endogenous AA to large quantities of 5-LO products as mononuclear phagocytes differentiate in the lung may equip them with the ability to mount an inflammatory response in the alveolar space.

Adenosine Triphosphate↗

PubMed1989

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. The intraperitoneal administration of 4 mmol/kg of BSO 30, 20, and 4 h prior to lung excision significantly depleted total lung glutathione as compared to saline-pretreated controls. This depletion of glutathione was associated with a significant attenuation of HPV in isolated perfused lungs but no alteration in pressor response to angiotensin II or KCl. In addition, hypoxia-associated LTC4 levels in lung homogenates were significantly lower in animals pretreated with BSO than in saline-pretreated controls. The specificity of the effects of BSO on lung leukotriene synthesis was examined by quantitating immunoreactive leukotrienes produced by unstimulated and ionophore A23187-stimulated parenchymal lung fragments, lonophore stimulation of lung fragments from BSO-pretreated rats produced 76 +/- 12.2% as much LTC4 and 127 +/- 22.3% as much leukotriene B4 as did fragments from saline-pretreated rats. Our data demonstrating that GSH depletion caused parallel reductions in both HPV and hypoxia-associated lung LTC4 levels are therefore consistent with the hypothesis that sulfidopeptide leukotrienes are involved in this pressor response in the rat.

Animals↗