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(1/1985) Association of the inflammatory state in active juvenile rheumatoid arthritis with hypo-high-density lipoproteinemia and reduced lipoprotein-associated platelet-activating factor acetylhydrolase activity.

OBJECTIVE: To investigate the relationship between the quantitative and qualitative abnormalities of apolipoprotein B (Apo B)- and Apo A-I-containing lipoproteins and between lipoprotein-associated platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with juvenile rheumatoid arthritis (JRA) as a function of the inflammatory state. METHODS: Twenty-six JRA patients and 22 age- and sex-matched control subjects with normal lipid levels participated in the study. Fourteen patients had active disease, and 12 had inactive disease. Plasma lipoproteins were fractionated by gradient ultracentrifugation into 9 subfractions, and their chemical composition and mass were determined. The PAF-AH activity associated with lipoprotein subfractions and the activity in plasma were also measured. RESULTS: Patients with active JRA had significantly lower plasma total cholesterol and high-density lipoprotein (HDL) cholesterol levels as compared with controls, due to the decrease in the mass of both the HDL2 and HDL3 subfractions. Patients with active JRA also had higher plasma triglyceride levels, mainly due to the higher triglyceride content of the very low-density lipoprotein plus the intermediate-density lipoprotein subfraction. The plasma PAF-AH activity in patients with active JRA was lower than that in controls, mainly due to the decrease in PAF-AH activity associated with the intermediate and dense low-density lipoprotein subclasses. The lipid abnormalities and the reduction in plasma PAF-AH activity were significantly correlated with plasma C-reactive protein levels and were not observed in patients with inactive JRA. CONCLUSION: This is the first study to show that patients with active JRA exhibit low levels of HDL2 and HDL3 and are deficient in plasma PAF-AH activity. These alterations suggest that active JRA is associated with partial loss of the antiinflammatory activity of plasma Apo B- and Apo A-I-containing lipoproteins.  (+info)

(2/1985) Interleukin-12 is synthesized by mesangial cells and stimulates platelet-activating factor synthesis, cytoskeletal reorganization, and cell shape change.

Preliminary studies indicate the involvement of interleukin (IL)-12 in experimental renal pathology. In the present study, we evaluated whether cultured glomerular mesangial cells are able to produce IL-12 and whether IL-12 may regulate some of their functions, including the cytoskeletal reorganization, the change in cell shape, and the production of platelet-activating factor (PAF). The results obtained indicate that pro-inflammatory stimuli, such as tumor necrosis factor-alpha and bacterial polysaccharides, induce the expression of IL-12 mRNA and the synthesis of the protein by cultured mesangial cells. Moreover, cultured mesangial cells were shown to bind IL-12 and to express the human low-affinity IL-12 beta1-chain receptor. When challenged with IL-12, mesangial cells produced PAF in a dose- and time-dependent manner and superoxide anions. No production of tumor necrosis factor-alpha and IL-8 was observed. Moreover, we demonstrate that IL-12 induced a delayed and sustained shape change of mesangial cells that reached its maximum between 90 and 120 minutes of incubation. The changes in cell shape occurred concomitantly with cytoskeletal rearrangements and may be consistent with cell contraction. As IL-12-dependent shape change of mesangial cells was concomitant with the synthesis of PAF, which is known to promote mesangial cell contraction, we investigated the role of PAF using two chemically different PAF receptor antagonists. Both antagonists inhibited almost completely the cell shape change induced by IL-12, whereas they were ineffective on angiotensin-II-induced cell shape change. In conclusion, our results suggest that mesangial cells can either produce IL-12 or be stimulated by this cytokine to synthesize PAF and to undergo shape changes compatible with cell contraction.  (+info)

(3/1985) Glutamate receptor signaling interplay modulates stress-sensitive mitogen-activated protein kinases and neuronal cell death.

Glutamate receptors modulate multiple signaling pathways, several of which involve mitogen-activated protein (MAP) kinases, with subsequent physiological or pathological consequences. Here we report that stimulation of the N-methyl-D-aspartate (NMDA) receptor, using platelet-activating factor (PAF) as a messenger, activates MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase, in primary cultures of hippocampal neurons. Activation of the metabotropic glutamate receptor (mGluR) blocks this NMDA-signaling through PAF and MAP kinases, and the resultant cell death. Recombinant PAF-acetylhydrolase degrades PAF generated by NMDA-receptor activation; the hetrazepine BN50730 (an intracellular PAF receptor antagonist) also inhibits both NMDA-stimulated MAP kinases and neuronal cell death. The finding that the NMDA receptor-PAF-MAP kinase signaling pathway is attenuated by mGluR activation highlights the exquisite interplay between glutamate receptors in the decision making process between neuronal survival and death.  (+info)

(4/1985) Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma.

Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor.  (+info)

(5/1985) In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome.

In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 microl drops compared with culture in groups of 10 in 10 microl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression.  (+info)

(6/1985) PAF binding to a single receptor in corneal epithelium plasma membrane.

PURPOSE: To study the binding characteristics and the expression of platelet-activating factor receptors (PAF-R) in corneal epithelium to elucidate the site of action of PAF. METHODS: Binding of [3H]PAF was investigated in subcellular fractions of the epithelia of bovine corneas and in membranes from cultured rabbit corneal epithelial cells. Dose-response inhibition curves of [3H]PAF-specific binding were generated using increasing concentrations of several PAF-R antagonists. RNA from rabbit corneal epithelial cells was probed for PAF-R expression by reverse transcription-polymerase chain reaction (RT-PCR) with specifically designed degenerated primers. RESULTS: Scatchard analysis showed a high-affinity binding site in bovine and rabbit corneal epithelium. The dissociation constant (Kd) and the maximum binding sites (Bmax) in a bovine membrane preparation and similar rabbit fraction were 0.77+/-0.03 nM and 180+/-21 femtomoles/mg protein and 4.3 nM and 1.3 picomoles/mg protein, respectively. Specific PAF-binding sites were found in bovine preparations enriched in plasma membranes with a Kd = 69.6 pM and Bmax = 80 femtomoles/mg protein; no specific binding was found in nuclei or microsomal fractions. RT-PCR of rabbit corneal epithelium generated a single product of the predicted size (478 bp). The deduced amino acid sequence of the purified PCR product was 87% homologous to human PAF-R. The hetrazepines BN 50727 and BN 50730 and the PAF structural analogues CV 3988 and CV 6209 competitively inhibited [3H]PAF binding to corneal epithelium with similar potency. WEB 2086 BS was two orders of magnitude less active in antagonizing PAF binding. CONCLUSIONS: Corneal epithelium contains a single population of receptors localized in the plasma membrane. PAF antagonists exert their actions by blocking this PAF-R. The partial sequence deduced in rabbit corneal PAF-R show a higher homology to the human PAF-R.  (+info)

(7/1985) Endogenous platelet-activating factor is critically involved in effector functions of eosinophils stimulated with IL-5 or IgG.

Eosinophil activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of allergic diseases. Eosinophils are activated by various classes of secretagogues, such as cytokines (e.g., IL-5), lipid mediators (e.g., platelet-activating factor (PAF)), and Ig (e.g., immobilized IgG). However, do these agonists act directly on eosinophils or indirectly through the generation of intermediate active metabolites? We now report that endogenous PAF produced by activated eosinophils plays a critical role in eosinophil functions. Human eosinophils produced superoxide when stimulated with immobilized IgG, soluble IL-5, or PAF. Pretreating eosinophils with pertussis toxin abolished their responses to these stimuli, suggesting involvement of a metabolite(s) that acts on G proteins. Indeed, PAF was detected in supernatants from eosinophils stimulated with IgG or IL-5. Furthermore, structurally distinct PAF antagonists, including CV6209, hexanolamine PAF, and Y-24180 (israpafant), inhibited IgG- or IL-5-induced superoxide production and degranulation. Previous reports indicated that exogenous PAF stimulates eosinophil eicosanoid production through formation of lipid bodies. We found in this study that IgG or IL-5 also induces lipid body formation and subsequent leukotriene C4 production mediated by endogenous PAF. Finally, inhibition of cytosolic phospholipase A2, one of the key enzymes involved in PAF synthesis, attenuated both PAF production and effector functions of eosinophils. These findings suggest that endogenous PAF plays important roles in eosinophil functional responses to various exogenous stimuli, such as cytokines and Igs. Therefore, inhibition of PAF synthesis or action may be beneficial for the treatment of eosinophilic inflammation.  (+info)

(8/1985) Mepyramine inhibits platelet activating factor-induced rabbit platelet aggregation: role of intracellular histamine.

AIM: To study the possible role of intracellular histamine (HA) in platelet activating factor (PAF)-induced platelet activation. METHODS: Washed rabbit platelet suspension was used to test the inhibitory effect of mepyramine (Mep, an H1 receptor antagonist) on PAF-induced platelet aggregation. The thromboxane B2 (TXB2) generation was measured by radioimmunoassay and the intracellular calcium ([Ca2+]i) concentration was determined by the specific fluorescence indicator Fura-2. RESULTS: Mep > 100 mumol.L-1 generated a concentration-dependent inhibition on PAF-induced aggregation, with an IC50 value of 162 (95% confidence limits: 114-232 mumol.L-1). Cimetidine, an H2 receptor antagonist, even up to 400 mumol.L-1 had no effect on it. Exogenous HA (10 mumol.L-1) and H1 receptor agonist, 2-thiazolylethylamine had no energetic effect. alpha-Fluoromethylhistidine, an inhibitor of histidine decarboxylase, did not inhibit platelet responses. However, in platelets permeabilized with saponin (8-10 mg.L-1), exogenous HA attenuated the inhibitory effect of Mep to about 50% at a concentration of 50 mumol.L-1. Preincubation of platelets with Mep (100 or 200 mumol.L-1) resulted in an inhibition on TXB2 generation and [Ca2+]i elevation induced by PAF. CONCLUSION: Platelets activated by PAF is associated with an intracellular HA synthesis and release via a common pathway of TXB2 generation and the rise of [Ca2+]i.  (+info)