Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2.

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.

Sphingolipids and cholesterol modulate membrane susceptibility to cytosolic phospholipase A(2).

Modulation of cytosolic phospholipase A(2) (cPLA(2)) activity by sphingomyelin (SPH), ceramide (Cer), and cholesterol (Chol) was investigated in CHO-2B cells activated by the calcium ionophore A23187 and epinephrine. Chol depletion of CHO-2B cells by treatment with methyl-beta-cyclodextrin (5 mm) resulted in the inhibition of the release of arachidonic acid whereas the restoration of the level by Chol-loaded cyclodextrin relieved inhibition. Conversion of CHO-2B cellular SPH to Cer by Staphylococcus aureus sphingomyelinase enhanced endogenous cPLA(2) activation as well as uptake by cells of C2- and C6-ceramide analogs. These results were confirmed in vitro with purified human recombinant cPLA(2) acting on a model phospholipid substrate. The enzyme activity was inhibited by SPH but reactivated by Cer as well as by Chol added to glycerophospholipid liposomal substrates containing SPH. The results of this study, which combine in situ and in vivo experimental approaches, indicate that membrane microdomains enriched in SPH and Chol play a role in the modulation of the activity of cPLA2 and in arachidonic acid-derived mediator production.

Comparison of a new method for the direct and simultaneous assessment of LDL- and HDL-cholesterol with ultracentrifugation and established methods.

BACKGROUND: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated. METHODS: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation. RESULTS: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination. CONCLUSIONS: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes.

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.

Differential stimulation of cytosolic phospholipase A2 by bradykinin in human cystic fibrosis cell lines.

Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the deltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. Immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A2 (cPLA2) was found in these cells and bradykinin increased its binding to membranes of deltaF508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-1). The expression of G alpha(q)/11 protein was also increased in deltaF508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA2 to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in deltaF508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA2 phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA2 to membranes after bradykinin stimulation still occurred in CFT2 cells (deltaF508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA2 anchoring to membranes. The defective processing of deltaF508 CFTR seems to increase cPLA2 stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The deltaF508 mutation of CFTR appears to increase the stimulation of cPLA2 by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed deltaF508 CFTR, inhibit cPLA2 stimulation. The greater reactivity of deltaF508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.

G proteins as biological targets for anti-allergic drugs?

The inhibiting effect of the H1 antihistamine cetirizine on the release of mediators (LTB4, arachidonic acid and phospholipase A2) was measured in different cells in vitro (human PMN, deltaF508 cells, chinese hamster ovary cells and rabbit chondrocytes) using different agonists (fMLP, NaF, calcium ionophore A 23187, bradykinin, adrenaline and IL-1). It was shown that physiological concentrations of the drug inhibited the release when activation of receptor-coupled G proteins was involved. By contrast, there was no inhibiting effect of cetirizine when the release was induced by a calcium ionophore which bypasses the G proteins coupled to cell membrane receptors.

Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.

Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity.

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

Secretory non-pancreatic phopholipase A2 in severe sepsis: relation to endotoxin, cytokines and thromboxane B2.

Circulatory secretory non-pancreatic phospholipase A2 (snp-PLA2) was measured prospectively at the onset (day 0) of severe sepsis in 52 patients as well as on day 1 and 2 in 25 patients, in order to answer two questions: 1) does the snp-PLA2 plasma concentration differ according to the type and severity of infection? 2) what is the relation between snp-PLA2 and other mediators involved in severe sepsis, such as endotoxin, cytokines (TNF alpha, IL-1 beta, IL-6) and thromboxane B2 (the stable metabolite of thromboxane A2)? On day 0, the snp-PLA2 circulatory level was 78 +/- 17 nmol/min/ml in patients with severe sepsis as compared to 3.5 +/- 2 nmol/min/ml in 40 healthy volunteers. There was no statistical difference according to the outcome, the presence of shock, or the type of infection on day 0. However, snp-PLA2 remained elevated or even increased in patients who ultimately died, while it decreased in survivors (p = 0.01 by ANOVA). The cytokine profiles during the 2-day follow-up were similar to that of snp-PLA2, but the differences were not statistically significant between survivors and non-survivors. No correlation was found between snp-PLA2 and other mediators for either initial or peak values.

Synergistic effect of interleukin-1 beta and tumor necrosis factor alpha on PGE2 production by articular chondrocytes does not involve PLA2 stimulation.

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL1), cause increased production of prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE2 production, and the activities of cytosolic phospholipase A2 (cPLA2), secreted phospholipase A2 (sPLA2), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA2, sPLA2, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE2 production. This synergy did not involve PLA2 stimulation, as there were no increases in AA release, cPLA2 and sPLA2 activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE2 production in articular chondrocytes. As sPLA2 and cPLA2 do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.

[Coupling of seven transmembrane domains receptors cytosolic PLA2: role of G proteins and protein kinases]. / Couplage des récepteurs à sept segments transmembranaires/PLA2 cytosolique: rôle des protéines G et des proteines kinases.

Cytosolic phospholipase A2 is a constitutive and ubiquitous enzyme. Although its role in the early production of lipid mediators is well established, the mechanisms leading to its activation and its place in the signal transduction pathways triggered by G-protein-coupled receptors is still unclear. Two main mechanisms, have been involved in its activation: its translocation by the increase of intracellular calcium allowing access to its phospholipidic substrate, and its phosphorylation by serine/threonine protein kinases such as protein kinase C or MAP kinases. However these two mechanisms do not fully explain the activation of cytosolic phospholipase A2. The irreversible association to membranes observed after receptor stimulation suggests that a still unknown anchoring mechanism might be involved. The elucidation of this anchoring might take place in the overall synthesis of distinct lipid mediators pools able to play an intracellular role of second messengers or an extracellular role of autocrine/paracrine mediators.

The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A.

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5'-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

Alpha 2 adrenergic receptor subtypes expressed in Chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21ras independent pathway.

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

Interleukin-1-induced prostaglandin E2 biosynthesis in human synovial cells involves the activation of cytosolic phospholipase A2 and cyclooxygenase-2.

Treatment of human synovial cells with interleukin-1 (IL-1) results in a large increase in the production of prostaglandin E2 (PGE2), a function in which the activation of phospholipase A2 (PLA2) is a key step. In order to identify the enzymes that are linked to IL-1-mediated arachidonate availability and subsequent PGE2 production, we have investigated the changes in gene expression of the 85-kDa cytosolic PLA2 (cPLA2), the 14-kDa secretory PLA2 (sPLA2) and the two forms of cyclooxygenase in human synoviocytes after stimulation with recombinant IL-1 beta. Northern-blot analysis revealed that both cPLA2 and cyclooxygenase-2 mRNA were progressively upregulated upon exposure to IL-1 for 5 hours and the glucocorticoid, dexamethasone, blocked the increased expression of these two genes. In contrast, IL-1-induced sPLA2 gene expression determined in the same cell samples was weak and most often rapid, and dexamethasone further stimulated it. In addition, IL-1 did not modify the levels of the constitutive cyclooxygenase-1. The cPLA2 and cyclooxygenase-2 enzymic activities are dependent upon de novo synthesis of mRNA and protein, since they were inhibited by actinomycin D and cycloheximide. Our data suggest that the IL-1-induced production of PGE2 in human synoviocytes can be attributed to the stimulation of both cPLA2 and cyclooxygenase-2. These enzymes may represent appropriate targets for selective blockade of prostanoid production in the inflammed joints.

Insulin-like growth factors counteract the effect of interleukin 1 beta on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes.

Interleukin 1 beta was found to stimulate arachidonic acid release, and the synthesis and secretion of type II phospholipase A2 by rabbit articular chondrocytes in vitro. Interleukin 1 beta had no effect on the level of cytosolic phospholipase A2 mRNA. Insulin-like growth factors, which help stabilize the cartilage matrix, reduced the effect of interleukin 1 beta on type II phospholipase A2 activity and mRNA level, and decreased the Interleukin 1 beta-stimulated arachidonic acid release to the basal values. This suggests that type II phospholipase A2 plays a key role in arachidonic acid release from rabbit articular chondrocytes and that insulin-like growth factors counteract the effect of interleukin 1 beta. They may therefore be considered as potential antiinflammatory agents.

Increase of bradykinin-stimulated arachidonic acid release in a delta F508 cystic fibrosis epithelial cell line.

Modification of chloride conductance by bradykinin in epithelial cells has been attributed to an activation of protein kinase A resulting from adenylcyclase stimulation by arachidonic acid cyclooxygenase products. The results presented here compare tracheal epithelial cell lines from one control and two cystic fibrosis patients which were immortalized by transfection with the SV40 large T oncogene. The three cell lines presented the same arachidonic acid content, turnover and mobilisation under basal conditions. Bradykinin stimulated the release of arachidonic acid and the synthesis of cyclooxygenase derivatives (mainly PGE2). The cell line from the cystic fibrosis patient bearing a phenylalanine 508 deletion, which is the major form of the disease, showed a higher bradykinin-induced arachidonic acid release than either control cells or cells from a patient presenting a minor form of the disease. This higher sensitivity suggests a dysregulation of phospholipase A2 stimulation in cystic fibrosis cells and was confirmed on non-immortalized tracheal epithelial cells in primary culture and on skin fibroblasts from patients bearing the same mutation. This defect is associated with a potentiation of cholera toxin pretreatment on cAMP content of delta F508 cell line. The impaired control of arachidonic acid release cannot be attributed to an increased number of bradykinin binding sites, since this increase was similar in the two cystic fibrosis cell lines.

Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages. Differential receptor/G-protein interaction assessed by pertussis and cholera toxins.

The involvement of guanine-nucleotide-binding regulatory proteins (G proteins) in the regulation of arachidonic-acid release induced by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or platelet-activating factor (PAF) was examined in guinea-pig alveolar macrophages. We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet-Leu-Phe or PAF) and of GTP (or GTP[S]). Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic-acid release and of the labeling of a 40-kDa protein in membranes incubated with [32P]NAD and pertussis toxin. fMet-Leu-Phe, but not PAF, allows the ADP-ribosylation of a 40-KDa protein by cholera toxin in the presence of Mg2+. This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin. The 40-kDa protein ADP-ribosylated seems to be alpha i1 and/or alpha i2. Stimulation of GTPase activity by fMet-Leu-Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin. Prior treatment of alveolar macrophages with cholera toxin, which ADP-ribosylates Gs, inhibits PAF-stimulated and fMet-Leu-Phe-stimulated arachidonic-acid release to the same extent, via a cAMP-protein-kinase-A cascade. The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet-Leu-Phe and PAF is associated with a strong increase of in-vitro [32P]NAD labeling of Gi proteins either by pertussis or by cholera toxin. This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein-phosphatase inhibitor, suggesting the involvement of protein-kinase A in this process. In conclusion, our results demonstrate that fMet-Leu-Phe and PAF receptors interact differently with Gi1/2 proteins in guinea-pig alveolar macrophages. Gi1/2 proteins are a possible target of the cross-regulation of arachidonic-acid release by a Gs-mediated pathway.