Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils

In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.     

Mechanisms involved in the bidirectional effects of protein kinase C activators on neutrophil responses to leukotriene B4

Three protein kinase C (PKC) activators (PMA, mezerein, and a diacylglycerol) had bidirectional effects on human polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4. Lower concentrations of the three agents enhanced, whereas higher concentrations inhibited, release of lysozyme and beta-glucuronidase stimulated by the arachidonic acid metabolite. Contrastingly, the activators inhibited but never enhanced LTB4-induced Ca2+ transients. We examined the causes for these varying effects. Each PKC activator reduced PMN specific binding of [3H]LTB4. Scatchard analyses revealed that PMA (greater than or equal to 0.16 nM) decreased the number of high affinity LTB4 receptors. The receptor losses correlated closely with inhibition of Ca2+ transients. PMN pretreated with 0.5 nM PMA for 5 min retained approximately 50% of their high affinity LTB4 receptors. These cells responded to 10 nM LTB4 with reduced but still substantial rises in cytosolic Ca2+, enhanced PKC mobilization, and increased granule enzyme release. The latter two effects appeared calcium-dependent because sequential exposure to PMA and LTB4 did not synergistically stimulate PKC mobilization or degranulation in PMN that were: 1) Ca2(+)-depleted; 2) challenged with 5 nM PMA; or 3) treated with LTB4 for 5 min before PMA. Each of the latter treatments completely interfered with the extent or timing of LTB4-induced Ca2+ transients. Accordingly, we suggest that the response-specific, bidirectional effects of PKC activators on LTB4 result from two opposing mechanisms. First, PKC activators down-regulate LTB4 high affinity receptors and thereby reduce those PMN responses that are not elicited by activated PKC (i.e., Ca2+ transients). Second, LTB4, by elevating cytosolic Ca2+, increases the amount of PKC mobilized by PKC activators and thereby promotes PKC-dependent responses (e.g., degranulation). The two mechanisms may be pertinent to the bidirectional effects of PKC activators on various other agonists. Furthermore, PKC, by down-regulating receptors, may serve as a physiologic stop signal for terminating function and producing a poststimulatory state of desensitization.     

Enhanced arachidonic acid metabolism and human neutrophil migration in asthma

In stable state asthmatic patients (AP) without any airway obstruction, the capacity of peripheral blood polymorphonuclear neutrophils (PMN) to produce 5-lipoxygenase metabolites and to migrate, was investigated and compared with the response in healthy subjects (HS). After calcium-ionophore A23187 stimulation, PMN from AP and HS produced LTB4, its hydroxylated derivatives: omega-OH-and omega-CO2H-LTB4) (omega-LTB4, i.e 6-trans-LTB4 and 5,6-diHETE isomers, and 5-HETE. We found an increase in LTB4 (+59%), omega-LTB4 (+39%), 6-trans-LTB4 (+128%), and free 5-HETE (+63%) generation of AP as compared with HS. Unstimulated migration was enhanced in AP (122 +/- 27 PMN/10 high power fields (hpf) in AP versus 74 +/- 25 PMN/10 hpf in HS, p less than 0.025) and suggested a greater capacity of PMN from AP to migrate. This was confirmed by the PAF-induced chemotaxis studies which showed, in AP, a greater PAF-sensitivity of PMN (10(-6) M versus 10(-5) M in HS) and a greater chemotaxis response (600 +/- 50 PMN versus 200 +/- 35 PMN in HS). In AP, we compared the capacity of PMN to generate LTB4 and 5-HETE with their capacity to migrate. We found an inverse correlation (r = 0.86, p less than 0.007) of intracellular free 5-HETE with chemotaxis to PAF.     

Platelet activating factor and leukotriene B4 induce hyperpolarization of human endothelial cells but depolarization of neutrophils

We studied one expression of cell activation in neutrophils (PMN) and endothelial cells (EC), membrane potential changes [assessed by the fluorescent dye, di-C-O5(3)]. Human neutrophils responded with depolarization after exposure to fMLP, LTB4, A23187, PAF and PMA. In contrast, only PAF and LTB4 induced membrane potential changes in human umbilical vein EC, which responded with increased fluorescence, possibly indicating membrane hyperpolarization. These discordant responses may reflect processes of significance for interactions between EC and PMN.     

Enhanced esterification process of 5-hydroxyeicosatetraenoic acid (5-HETE) in PMN from asthmatic patients.

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory properties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min, total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 +/- 3% versus 47 +/- 2% of the total synthesis, p less than 0.005), whereas intracellular free 5-HETE was decreased (13 +/- 3% versus 37 +/- 4%, p less than 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.     

Prostaglandin binding sites in human polymorphonuclear neutrophils

Prostaglandin (PG) E2 (greater than or equal to 1.6 nM) and PGD2 (greater than or equal to 16 nM) inhibited polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4 and platelet-activating factor (PAF) whereas PGF2 alpha was bioinactive. [3H]PGE2 and [3H]PGD2 bound to PMN and isolated, plasmalemma-enriched PMN membranes. Binding was time-dependent, specific, saturable, and reversible. Competitive studies indicated that the two PGs bound to distinctly different sites. PMN had high (Kd = 1 nM; Rt = 150/cell) and low (Kd = 100 nM; Rt = 5800/cell) affinity PGE2 binding sites. Only a single type of PGD2 binding site (Kd = 13 nM; Rt = 5100/cell) was detected. We conclude that PGE2 and PGD2 bind to their respective, plasmalemmal receptors to attenuate PMN function. The PGs may act as endogenous stop signals to limit the action of concurrently formed excitatory signals, eg., LTB4 and PAF.     

Intracellular retention of the 5-lipoxygenase pathway product, leukotriene B4, by human neutrophils activated with unopsonized zymosan

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.     

On the relationship between leukotriene and lipoxin production by human neutrophils: evidence for differential metabolism of 15-HETE and 5-HETE

Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB4 and its omega-oxidation products as well as LXA4, LXB4, and 7-cis-11-trans-LXA4 from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB4 and its omega-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[3H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.     

Receptor-operated activation of polymorphonuclear leukocytes: different effects of NAP-1/IL-8 and fMet-Leu-Phe or C5a.

We examined the production of PAF and LTB4 by PMN in response to NAP1/IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-1/IL-8 does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-1/IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAP1/IL-8. Our data reveal a possible autocrine role of PAF and LTB4 in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or C5a, but not NAP-1/IL-8.     

Leukotriene B4 induced hyperadhesiveness of endothelial cells for neutrophils

Leukotriene B4 (LTB4) induced a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC), leading to increased binding of neutrophil granulocytes (PMN). The effect of LTB4 was more rapidly emerging and transient than responses to platelet activating factor (PAF), thrombin and phorbol myristate acetate (PMA). At 0.1 microM of LTB4, it was comparable to hyperadhesiveness induced by 1 U/ml of thrombin, but less than that conferred by 0.1 microM of PAF and PMA. The adherence response to LTB4 was specific since the structural analogue 5S,12S-diHETE, which lacks PMN-stimulating effects, failed to promote HUVEC adhesiveness.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

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.     

Enhanced esterification process of 5-hydroxyeicosatetraenoic acid (5-HETE) in PMN from asthmatic patients

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory porperties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min , total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 ± 3 % versus 47 ± 2 % of the total synthesis, p < 0.005), whereas intracellular free 5-HETE was decreased (13 ± 3 % versus 37 ± 4 %, p < 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.     

Inhibition of production of LTB4 and chemotactic agent from rat alveolar macrophages treated with t-butyl hydroperoxide is independent of ATP depletion

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.     

Alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do monocytes.

Alveolar macrophages release greater amounts of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) after A23187 stimulation than do blood monocytes. The mechanisms for this enhanced 5-lipoxygenase activity in alveolar macrophages are unknown. In these studies, we determined whether alveolar macrophages have greater amounts of the enzyme 5-lipoxygenase than do blood monocytes. We confirmed that alveolar macrophages released greater amounts of LTB4 after A23187 stimulation than did equivalent numbers of blood monocytes. In both the presence and absence of A23187, alveolar macrophages had greater amounts of immunoreactive 5-lipoxygenase, determined by Western analysis, on a per cell and a per protein basis than did blood monocytes. The amounts of 5-lipoxygenase enzyme in the cells roughly correlated with the amounts of LTB4 released by both types of cells. These observations suggest that A23187 stimulates alveolar macrophages to release greater amounts of LTB4 and 5-HETE than blood monocytes, in part, due to the greater amounts of 5-lipoxygenase.     

Arachidonic acid and 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid modulate human polymorphonuclear neutrophil activation by monocyte derived neutrophil activating factor

Exposure of human polymorphonuclear neutrophils (PMN) to human monocyte derived neutrophil activating factor(s) (NAF) resulted in a concentration-dependent extracellular release of granule constituents. NAF also induced the generation of 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [Leukotriene B4 (LTB4)] by PMNs which was enhanced in the presence of exogenous arachidonic acid (AA). In contrast to its enhancing effect on LTB4 production, AA inhibited NAF-stimulated PMN degranulation. 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a product of the 15-lipoxy-genation of AA in PMNS, caused a concentration-dependent suppression of degranulation and LTB4 generation by PMNs in contact with NAF. 15-HETE also inhibited the rise in cytosolic-free calcium [( Ca2+]i) observed in NAF activated PMNs. These data suggest that AA and a 15-lipoxygenase product modulate the NAF-associated activation pathway in human PMNs.     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.