Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.     

Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase

Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.     

Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria

OBJECTIVE: To investigate the role of phospholipase during the activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by peritoneal dialysis effluent (PDE). DESIGN: Examine the action of 4-hour dwell PDE upon phospholipase activation in the circulating neutrophils obtained from healthy individuals. RESULTS: We have previously reported that PDE stimulated superoxide release by the NADPH oxidase of human neutrophils and primed the response to the bacterial peptide, fMLP (fMetLeuPhe). To elucidate the biochemical mechanisms underlying these observations, we have examined the roles of phospholipases (PL) C, D, and A2, whose activation causes the release of a range of intracellular secondary messengers. Following fMLP stimulation, we observed a rapid activation of both PLC and PLD as well as a small but nonsignificant increase in PLA2 activity. Peritoneal dialysis effluent alone failed to stimulate either PLC or PLD, while pre-incubation with PDE had no affect upon fMLP-induced PLC and PLD activation. However, PDE caused a small but nonsignificant increase in PLA2 activity (which was comparable to that observed with fMLP) and primed the fMLP-induced response. In common with a role for PLA2 and the subsequent release of arachidonic acid (AA), we have demonstrated dose-dependent inhibition of PDE-induced superoxide release by the PLA2 inhibitor mepacrine, as well as activation and priming of the fMLP-induced superoxide generation by AA. CONCLUSIONS: These results imply that PDE-induced NADPH-oxidase activation and priming in human neutrophils is mediated via a PLA2-dependent but PLC- and PLD-independent mechanism.     

Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2-) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2- production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2- production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2-, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.     

Cross talk between substance P and melittin-activated cellular signaling pathways in rat lactotroph-enriched cell cultures

We have investigated the possible interaction (cross talk) between the phospholipase A2 (PLA2) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [3H]arachidonic acid ([3H]AA) from [3H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKC alpha and beta immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Melittin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA2 inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [3H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [3H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [3H]AA production was not different from control, whereas SP still displayed [3H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [3H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [3H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of 3H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA2 pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes alpha and beta, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA2 pathway at a level at or beyond PLA2, and this effect is mediated, in part, through PKC alpha and beta species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA2 through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.     

Survival and water-balance characteristics of unfed adult Amblyomma cajennense (Acari: Ixodidae)

The specific type of phospholipase A2 (PLA2) involved in formation of leukotriene B4 (LTB4) and platelet activating factor (PAF) in inflammatory cells has been controversial. In a recent report we characterized activation of the 'cytosolic' form of PLA2 (cPLA2) in human neutrophils (PMN) permeabilized with Staphylococcus aureus alpha-toxin under conditions where the secretory form of PLA2 (sPLA2) was inactive. In the current study, generation of both LTB4 and PAF in porated PMN are demonstrated. PMN, prelabeled with [3H]arachidonic acid (3H-AA, to assess AA release and LTB4 production) or with 1-O-[9',10'-3H]hexadecyl-2-lyso-glycero-3-phosphocholine (3H-lyso-PAF, for determination of lyso-PAF and PAF formation), were permeabilized with alpha-toxin in a 'cytoplasmic' buffer supplemented with acetyl CoA. Maximum production of both PAF and LTB4 required addition of 500 nM Ca2+, G-protein activation induced with 10 microM GTP gamma S, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 microM); LTB4 production was confirmed by radioimmunoassay. Removal of acetyl CoA from the system had little effect on LTB4 generation but blocked PAF production with a concomitant increase in lyso-PAF formation LTB4 and PAF production occurred in parallel over time and at differing ATP and Ca2+ concentrations. Further work demonstrated that: (i) maximum production of both inflammatory mediators required a hydrolyzable form of ATP; (ii) blocking phosphorylation with staurosporin inhibited production of both; (iii) the reducing agent, dithiotreitol, had little affect on LTB4 formation but slightly enhanced PAF generation. This study clearly shows that cPLA2 activation can provide precursors for both LTB4 and PAF, that maximum PAF and LTB4 formation occur under conditions that induced optimal cPLA2 activation, that a close coupling between LTB4 and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB4 and PAF generation in the permeabilized PMN system.     

Dietary alpha-tocopherol supplementation on antioxidant defenses after in vivo iron overload in rats

In the present study we have examined the effects and mechanisms of endothelin-1 (ET-1) on arachidonic acid (AA) release and prostaglandin (PG) synthesis in human ciliary muscle (HCM) cells. ET-1 stimulated AA release in a time (t1/2=1.5 min) and concentration-dependent (EC50=5 nM) manner, which is primarily mediated through the ETA receptor subtype. The AA liberated by ET-1 appears to derive mainly from the phosphoinositides and phosphatidylcholine. Our data show that phospholipase A2 (PLA2), but not phospholipase C (PLC), plays an important role in ET-1-induced AA release. This conclusion is supported by the following findings: (1) ET-1-evoked AA release was inhibited by the PLA2 inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE2 was inhibited by the cyclooxygenase inhibitors in the following order: Indomethacin>naproxen >ibuprofen>NS-398>aspirin. (2) The phorbol ester, PDBu, an activator of protein kinase C, potentiated ET-1-induced AA release by 39%, but inhibited that of inositol phosphates formation by 62%. (3) Pretreatment of the labeled cells with isoproterenol lowered ET-1-induced inositol phosphates production, but had no effect on AA release. (4) U71322, a PLC inhibitor, inhibited ET-1-induced inositol phosphates production, but had no effect on that of AA release. (5) Pretreatment of the cells with pertussis toxin (0.1 microg ml-1) attenuated the stimulatory effects of ET-1 on AA release and PGE2 formation. These data demonstrate that ET-1 is a potent agonist for AA release and PG synthesis in HCM cells, and that PLA2, but not PLC, plays an important role in ET-1-induced AA release and PG synthesis. In ciliary muscle, AA and its metabolites play important roles in intracellular signalling, modulation of physiological processes, and regulation of intraocular pressure.     

Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.     

Regulation of CD11b/CD18 expression in human neutrophils by phospholipase A2

Recent evidence suggests that phospholipase A2 (PLA2)-derived lipid mediators may regulate a number of neutrophil responses including degranulation and adhesion. In view of the potential role of PLA2 in stimulus-secretion coupling, we examined the relationship between PLA2 activation and the surface expression of CD11b/CD18 (MAC-1) in human polymorphonuclear leukocytes (hPMNL), including the functional consequences of PLA2 inactivation on MAC-1-dependent adhesion. The selective inhibition of PLA2 by the marine natural products manoalide (MLD) and scalaradial (SLD) blocks [3H]arachidonic acid (AA) release in calcium ionophore A23187-stimulated neutrophils, and also inhibits secretion of specific and azurophilic granule constituents. Additional studies demonstrate that MLD, SLD, and other less potent PLA2 inhibitors such as 4-bromophenacylbromide and nordihydroguiaretic acid inhibit the surface expression of MAC-1 (IC50: MLD, 0.33 microM; SLD, 0.23 microM; 4-bromophenacylbromide, 2.8 microM; NDGA, 3.5 microM) at concentrations similar to those at which they inhibit [3H]AA release. Inhibitors of cyclooxygenase, 5-lipoxygenase, protein kinase C, or calcium channel antagonists have no effect on MAC-1 expression. PLA2 inactivation also prevents MAC-1 up-regulation in hPMNL stimulated with FMLP, IL-8, TNF-alpha, PMA, or platelet activating factor. In FMLP-stimulated hPMNL, under conditions in which no secondary granule constituents are secreted, MAC-1 and alkaline phosphatase up-regulation from intracellular granules is inhibited by MLD and SLD. Functional assays also demonstrate that MLD and SLD block MAC-1-dependent adhesion of activated neutrophils to keyhole limpet hemocyanin at concentrations that block the surface expression of MAC-1. [3H]AA release and MAC-1 expression in MLD and SLD-treated hPMNL could be recovered in the presence of 1 mM hydroxylamine in a time-dependent fashion, consistent with reported data that MLD and SLD inactivate PLA2 through Schiff base formation. In summary, these data emphasize the role of PLA2 as a key regulator of MAC-1 expression in models of neutrophil adhesion.     

Cloricromene inhibits leukotriene formation by human polymorphonuclear leucocytes by suppressing arachidonate release from membrane phospholipids

Cloricromene, an antithrombotic agent known to inhibit the release of arachidonic acid (AA) in stimulated human platelets, was tested for its effects on arachidonate release and metabolism in human polymorphonuclear leucocytes (PMNs). Cloricromene dose-dependently suppressed the release of leukotriene B4 (LTB4), as assessed by radioimmunoassay, from both isolated PMNs and human whole blood stimulated with the calcium ionophore A23187 or with serum-treated zymosan (STZ). The inhibitory effect was higher when the concentration of the stimulating agent was weaker. Cloricromene also inhibited dose-dependently the liberation of LTB4, LTC4, LTD4 and 5-hydroxy-6,8,11,14-eicosatraenoic acid as assessed by HPLC in the supernantant of A23187-stimulated PMNs. Finally, the drug was able to suppress the release of [3H]AA from purified human PMNs prelabeled with the radioactive fatty acid and stimulated with either A23187 or with STZ. The A23187-induced decrease in the radioactivity of phosphatidylinositol, the phospholipid class mainly involved in AA release in stimulated PMNs, was also inhibited by cloricromene. Cloricromene suppresses leukotriene formation in human PMNs by reducing AA release from membrane phospholipids, possibly through interference with phospholipase A2 activation; this activity may contribute to the leucocyte-inhibitory effects reported previously for cloricromene.     

Thoracoabdominal aortic aneurysm repair in patients with single kidney

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.     

Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.     

Arachidonic acid protects against hypoxic injury in rat proximal tubules

Free fatty acids (FFA) and lysophospholipids accumulate during hypoxia (H) in rat proximal tubular epithelial cells partly as a result of increased phospholipase A2 (PLA2) activity. The role of FFA in mediating hypoxic injury and modulating PLA2 activity is not clear. In the present study, the effect of several FFA including arachidonic acid (AA, 20:4) on hypoxia-induced injury and PLA2 activity was assessed in freshly isolated rat proximal tubules. Hypoxia (H) was induced in the presence of either an unsaturated free fatty acid (uFFA) [AA or linoleic acid (LA, 18:2)] or a saturated FFA (sFFA) [palmitic (PA, 16:0) or myristic acid (MA, 14:0)]. Cell membrane injury was assessed by measuring lactate dehydrogenase release (LDH). AA markedly reduced LDH release during hypoxia in a dose dependent manner, while sFFA had no protective effect. LA showed similar protection to that observed with AA. AA did not affect buffer calcium concentration, buffer pH, intracellular pH or intracellular calcium concentration. Neither inhibiting the cyclooxygenase pathway with indomethacin, nor the lipoxygenase pathway with nordihydroguaiaretic acid (NDGA) had any effect on the AA observed cytoprotection. In vitro PLA2 activity in the control tubular extracts was compared to that following addition of AA or PA. PLA2 activity decreased significantly with AA but not with PA in a dose dependent manner. These data suggest that: (1) AA protects against hypoxic injury in rat proximal tubules. (2) This cytoprotection is not specific for AA and other uFFA have a similar effect. (3) AA significantly inhibits PLA2 activity, (4) AA induced cytoprotection may be related to a negative feedback inhibition of PLA2 activity.     

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.     

Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.     

Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells

This study investigated the signal transduction mechanisms of angiotensin-(1-7) [Ang-(1-7)]- and Ang II-stimulated arachidonic acid (AA) release for prostaglandin (PG) production in rabbit aortic vascular smooth muscle cells. Ang II and Ang-(1-7) enhanced AA release in cells prelabeled with [3H]AA. However, 6-keto-PGF1 alpha synthesis produced by Ang II was much less than that caused by Ang-(1-7). In the presence of the lipoxygenase inhibitor baicalein, Ang II enhanced production of 6-keto-PGF1 alpha to a greater degree than Ang-(1-7). Angiotensin type (AT)1 receptor antagonist DUP-753 inhibited only Ang II-induced [3H]AA release, whereas the AT2 receptor antagonist PD-123319 inhibited both Ang II- and Ang-(1-7)-induced [3H]AA release. Ang-(1-7), receptor antagonist D-Ala7-Ang-(1-7) inhibited the effect of Ang-(1-7), but not of Ang II. In cells transiently transfected with cytosolic phospholipase A2 (cPLA2), mitogen-activated protein (MAP) kinase or Ca(++)-/cal-modulin-dependent protein (CAM) kinase II antisense oligonucleotides, Ang-(1-7)- and Ang II-induced [3H]AA release was attenuated. The CaM kinase II inhibitor KN-93 and the MAP kinase kinase inhibitor PD-98059 attenuated both Ang-(1-7)- and Ang II-induced cPLA2 activity and [3H]AA release. Ang-(1-7) and Ang II also increased CaM kinase II and MAP kinase activities. Although KN-93 attenuated MAP kinase activity, PD-98059 did not affect CaM kinase II activity. Both Ang II and Ang-(1-7) caused translocation of cytosolic PLA2 to the nuclear envelope. These data show that Ang-(1-7) and Ang II stimulate AA release and prostacyclin synthesis via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn, via MAP kinase activation, enhances cPLA2 activity and release of AA for PG synthesis.     

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.     

Diltiazem compared with placebo in the prevention of myocardial ischemia during non-cardiac surgery

OBJECTIVE: Because eosinophils likely play important roles in the pathophysiology of allergic diseases, specific inhibitors of eosinophils may be desirable to treat such diseases. To evaluate the capacity of a novel compound, sulochrin, as an inhibitor of eosinophilic inflammation, we examined the effects of this compound on various effector functions of eosinophils. MATERIALS AND METHODS: We examined the effects of sulochrin on degranulation of human eosinophils stimulated with platelet-activating factor (PAF) or Sepharose 4B beads coated with secretory IgA (sIgA) or IgG. The effects of sulochrin on other effector functions of human eosinophils, including superoxide anion (O2-) production, leukotriene (LT) C4 release, and interleukin (IL)-8 production induced by sIgA-beads were also studied. Finally, using PAF and LTB4 as chemoattractants, we evaluated the potency of sulochrin to inhibit eosinophil migration in vitro and in vivo. RESULTS: Sulochrin inhibited EDN release by eosinophils stimulated with sIgA-beads. IgG-beads and PAF in a concentration-dependent manner; IC50 values were 0.75 microM, 0.30 microM and 0.03 microM. Eosinophil O2- production, LTC4 release, and IL-8 production were also inhibited by sulochrin. Furthermore, PAF-induced chemotaxis of human eosinophils and LTB4-induced chemotaxis of guinea pig eosinophils were abolished by 1 microM of sulochrin. Finally, sulochrin potently inhibited LTB4-induced infiltration of eosinophils into the skin of guinea-pig in vivo. CONCLUSIONS: These results suggest that sulochrin is a potent inhibitor of various effector functions of eosinophils. Sulochrin and its derivatives may be useful in the development of therapeutic approaches for patients with allergic diseases.