Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: Requirement for a Giα-type G-protein

Burke, James R.; Davern, Lynda B.; Gregor, Kurt R.; Todderud, Gordon; Alford, Julie; Tramposch, Kenneth M.

doi:10.1016/s0167-4838(97)00085-x

Cited by 37 publications

(24 citation statements)

References 64 publications

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“…Cell Assays For measurements of the effects of cPLA 2 inhibitors on cellular arachidonate production, dibutyryl-cAMP-differentiated U937 cells were used according to the previously published procedure (33). In short, cells were preincubated with the inhibitors for 5 min at 37°C prior to stimulating with the chemotactic peptide, fMLP.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…In short, cells were preincubated with the inhibitors for 5 min at 37°C prior to stimulating with the chemotactic peptide, fMLP. After 5 min of stimulation, the reactions were terminated in an ice bath, and the mass of arachidonate determined by GC/electron capture of derivatized product (33).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…It has been shown that when these cells are stimulated with the chemotactic peptide fMLP, a robust release of arachidonate occurs. Moreover, this release is the result of the action of cPLA 2 (33). fMLP stimulation of these cells also leads to the activation of phospholipase C with production of inositol 1,4,5-trisphosphate (33).…”

Section: -(2ј-benzyl-4ј-chlorophenoxy)ethyl-n-dodecylamine Hydrochlomentioning

confidence: 99%

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Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Burke¹,

Witmer²,

Zusi

et al. 1999

Journal of Biological Chemistry

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Cytosolic phospholipase A 2 (cPLA 2 ) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA 2 using membranes isolated from [ 3 H]arachidonate-labeled U937 cells as substrate, 2-(2-benzyl-4-chlorophenoxy)ethyl-dimethyln-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC 50 ‫؍‬ 5 M). It was over 70 times more selective for the cPLA 2 as compared with the human nonpancreatic secreted phospholipase A 2 , and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6 -10 mol % of 1-palmitoyl-2-[1-14 C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K I * app ) was determined to be 0.097 ؎ 0.032 mol % versus an apparent dissociation constant for the arachidonatecontaining phospholipid of 0.3 ؎ 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA 2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller K I * app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [ 3 H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [ 3 H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

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Section: Enzyme Assaysmentioning

confidence: 99%

Section: Enzyme Assaysmentioning

confidence: 99%

Section: -(2ј-benzyl-4ј-chlorophenoxy)ethyl-n-dodecylamine Hydrochlomentioning

confidence: 99%

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Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Burke¹,

Witmer²,

Zusi

et al. 1999

Journal of Biological Chemistry

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“…In addition to adenylyl cyclase inhibition, receptors coupled to pertussis toxin-sensitive heterotrimeric G proteins (e.g., H 3 R) are known to stimulate phospholipase A 2 (PLA 2 ) via the Gα i subunit [6][7][8]. Furthermore, H 3 R couple to the MAPK cascade [9] which contributes to PLA 2 phosphorylation and stimulation of its catalytic activity [10].…”

Section: Introductionmentioning

confidence: 99%

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

Levi

Seyedi

Schäefer

et al. 2007

Biochemical Pharmacology

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We tested the hypothesis that the histamine H 3 -receptor (H 3 R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Gα imediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gβγ i -mediated activation of the MAPK-PLA 2 cascade, culminating in formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE 2 ). We report in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H 3 R-mediated attenuation of K + -induced NE exocytosis was prevented by MAPK and PLA 2 inhibitors, and by cyclooxygenase and EP 3 -receptor (EP 3 R) antagonists. Moreover, H 3 R activation resulted in MAPK phosphorylation in H 3 R-transfected SH-SY5Y neuroblastoma cells, and in PLA 2 activation and PGE 2 production in cardiac synaptosomes; H 3 R-induced MAPK phosphorylation was prevented by an anti-βγ peptide. Synergism between H 3 R and EP 3 R agonists (i.e., imetit and sulprostone, respectively) suggested PGE 2 may be a downstream effector of the anti-exocytotic effect of H 3 R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca 2+ -channel antagonist ω-conotoxin GVIA, and prevented by an anti-Gβγ peptide. Our findings suggest an EP 3 R Gβγ i -induced decrease in Ca 2+ influx through N-type Ca 2+ -channels is involved in PGE 2 / EP 3 R-mediated attenuation of NE exocytosis elicited by H 3 R activation. Conceivably, activation of the Gβγ i subunit of H 3 R and EP 3 R may also inhibit Ca 2+ entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H 3 R activation is cardioprotective. Thus, the uncovering of a novel H 3 R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.

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“…However, treatment with C 2 -ceramide had no effect on the forskolin-induced formation of cAMP. Mastoparan is a cationic peptide which selectively binds to the receptor-recognition site of heterotrimeric G-proteins (7,28 …”

Section: Effect Of C 2 -Ceramide On the Accumulation Of Camp Induced mentioning

confidence: 99%

C2-ceramide inhibits the prostaglandin E2-induced accumulation of cAMP in human gingival fibroblasts

et al. 2010

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Ceramide is generated by the hydrolysis of membrane sphingomyelin by sphingomyelinase and is implicated in multiple signaling pathways, including those regulating differentiation, inflammation and immune responses. Excess formation of prostaglandin E 2 (PGE 2 ) is thought to increase susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases. We investigated the inhibitory effect of C 2 -ceramide, a short-chain ceramide analog, on the PGE 2 -stimulated accumulation of cAMP in human gingival fibroblasts. In human gingival fibroblasts pre-treated with C 2 -ceramide for 18 h, the PGE 2 -stimulated accumulation of cAMP was reduced, but an inactive C 2 -ceramide analog had no such effect. The accumulation of cAMP induced by EP2 and EP4 receptor agonists (ONO-AE1-259 and ONO-AE1-329, respectively) was inhibited in cells treated with C 2 -ceramide. However, treatment with C 2 -ceramide had no effect on the expression of mRNAs encoding the EP2 and EP4 receptors. Accumulation of cAMP could be induced by cAMP-elevating agents (forskolin, isobutylmethylxanthine and mastparan) but was not reduced by treatment with C 2 -ceramide. These observations suggest that C 2 -ceramide attenuates PGE 2 receptor function and consequently inhibits the accumulation of cAMP in human gingival fibroblasts.Prostaglandins are lipid mediators that are involved in many physiological and pathophysiological processes. The excess formation of prostaglandin E 2 (PGE 2 ) has been described in a number of conditions characterized by increased susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases (8,17,25,29). PGE 2 mediates its biological functions via binding to four types of membrane-bound, G protein-coupled receptors, termed E prostanoid (EP)1 to EP4 (6, 22). EP receptors stimulated by ligand binding activate different signal transduction pathways. Activation of the EP1 receptor raises intracellular Ca 2+ levels. Activation of EP2 and EP4 receptors increases intracellular cAMP levels by activating adenylate cyclase via G s proteins. Activation of the EP3 receptor reduces or increases cAMP levels by activating inhibitory G (G i ) or stimulatory G (G s ) proteins depending on the particular splice variant expressed by the cell (15). In human gingival fibroblasts, PGE 2 inhibited the DNA synthesis and proliferation (1, 37), and downregulated intercellular adhesion molecule-1 expression by cAMP-dependent signaling pathways (24). Therefore, cAMP is thought to be the main intracellular second messenger for response to PGE 2 , and is the crucial modulator of the functional activity of inflammation. Ceramide, an intracellular second messenger in the sphingomyelin transmembrane signaling pathway, can be generated by the cleavage of sphingomyelin by sphingomyelinase or by de novo biosynthesis by ceramide synthase (14). Increases in intracellular ceramide have been reported in many

show abstract

Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: Requirement for a Giα-type G-protein

Cited by 37 publications

References 64 publications

Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

C2-ceramide inhibits the prostaglandin E2-induced accumulation of cAMP in human gingival fibroblasts

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