Oleoylethanolamide restores alcohol-induced inhibition of neuronal proliferation and microglial activity in striatum.

Previous findings demonstrate a homeostatic role for oleoylethanolamide (OEA) signaling in the ethanol-related neuroinflammation and behavior. However, extensive research is still required in order to unveil the effects of OEA on a number of neurobiological functions such as adult neurogenesis, cell survival and resident neuroimmunity that become notably altered by alcohol. Daily consumption of ethanol (10%) for 2 weeks (6.3 ± 1.1 g/kg/day during last 5 days) caused hypolocomotor activity in rats. This effect appears to rely on central signaling mechanisms given that alcohol increased the OEA levels, the gene expression of OEA-synthesizing enzyme Nape-pld and the number of PPARα-immunoreactive neurons in the striatum. Ethanol-related neurobiological alterations such as a reduction in the number of microglial cells expressing iNOS (a cytokine-inducible immune defense) and in adult neural stem/progenitor cell (NSPC) proliferation (phospho-H3 and BrdU) and maturation (BrdU/ß3-tubulin), as well as an increase in damage cell activity (FosB) and apoptosis (cleaved caspase 3) were also observed in the rat striatum. Pharmacological administration of OEA (10 mg/kg) for 5 days during ethanol exposure exacerbated ethanol-induced hypolocomotion and cell apoptosis in the striatum. Interestingly, OEA abrogated the impaired effects of ethanol on PPARα-positive cell population and NSPC proliferation and maturation. OEA also decreased astrocyte-related vimentin immunoreactivity and increased microglial cell population (Iba-1, iNOS) in the striatum. These results suggest that OEA-PPARα signaling modulates glial activation, cell apoptosis and NSPC proliferation and maturation in response to striatal-specific neurobiological alterations induced by prolonged ethanol intake in rats.

Phospholipase D1 expression analysis in relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic disorder resulting from destruction of the myelin or insulating covers of neurons in the central nervous system (CNS). Several lines of evidence suggest a role for immune response in the occurrence and progression of this disorder. Several disease-modifying agents (DMA) including ß-interferons (IFNß) are being used in MS patients in order to stop the disease at the early inflammatory stage, postpone disease progression and diminish future disability. Phospholipase D1 (PLD1) is a critical enzyme responsible for the making lipid second messenger phosphatidic acid. It has an established function in regulation of immune response. In the present study we have evaluated PLD1 transcript levels and plasma concentrations in 78 relapsing-remitting MS (RRMS) patients as well as 78 normal age- and sex-matched healthy subjects using real-time quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Significant PLD1 down-regulation has been observed in total MS patients compared with controls (P < 0.001) as well as IFN-ß responders (P = 0.034) and non-responders (P < 0.001) compared with controls, respectively. However, a significant up-regulation has been detected in IFN-ß responders compared with non-responders (P = 0.047). In both males and females groups, significant down-regulations have been detected in patients compared with controls (P = 0.014 and P = 0.002, respectively). The same results have been detected in PLD1 plasma concentrations. In conclusion, PLD1 transcripts in blood and its plasma concentrations can be used as putative biomarkers for evaluation of therapeutic responses to IFN-ß in RRMS patients. However, this result should be validated in future studies.

Regulation of glycosylphosphatidylinositol-anchored proteins and GPI-phospholipase D in a c-Myc transgenic mouse model of hepatocellular carcinoma and human HCC.

Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specific phospholipase D (GPI-PLD) in the pathogenesis of fatty liver disease and hepatocellular carcinoma (HCC). Given that c-Myc is frequently amplified in HCC, we investigated their regulation in a c-Myc transgenic disease model of liver cancer and HCC patient samples. Whole genome scans defined 54 significantly regulated genes coding for GPI-AP of which 29 and 14 were repressed in expression in transgenic tumors and steatotic human hepatocyte cultures, respectively, to influence lipid-mediated signal transduction, extracellular matrix and immunity pathways. Analysis of gene specific promoter revealed >95% to carry c-Myc binding sites thus establishing a link between c-Myc activity and transcriptional response. Alike, serum GPI-PLD activity was increased 4-fold in transgenic mice; however its tissue activity was reduced by 70%. The associated repression of the serine/threonine phosphatase 2A (PP2A), i.e. a key player of c-Myc proteolysis, indicates co-ordinate responses aimed at impairing tissue GPI-PLD anti-proliferative activities. Translational research identified >4-fold increased GPI-PLD serum protein expression though enzyme activities were repressed by 60% in NASH and HCC patients. Taken collectively, c-Myc influences GPI-AP signaling transcriptionally and posttranslational and represses GPI-AP anti-proliferative signaling in tumors. The findings broaden the perspective of molecular targeted therapies and disease monitoring.

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

BACKGROUND: Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. METHODS: The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. RESULTS: The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P <0.001), and serum levels of TNF-α (P <0.001) and interleukin-6 (P <0.001) after ADA medication, although plaque levels were comparable. Among a total of 495 protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P <0.01). CONCLUSION: These results suggest a beneficial effect of ADA therapy on the periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid.

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Statin therapy reduces serum levels of glycosylphosphatidylinositol-specific phospholipase D.

Statin therapy is associated with changes in low-density, very low-density, and high- density lipoprotein metabolism. The effect of statin therapy on a minor high-density lipoprotein particle containing glycosylphosphatidylinositol-specific phospholipase D has not been examined. Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) has been implicated in triglyceride metabolism. A double-blind, crossover design comparing the effect of simvastatin (80 mg) and atorvastatin (80 mg) on serum lipid and glycosylphosphatidylinositol-specific phospholipase D levels was conducted in 13 patients with low high-density lipoproteins. Both statins reduced cholesterol, triglycerides, and apolipoprotein B and significantly lowered serum glycosylphosphatidylinositol-specific phospholipase D levels (16%). This statin effect seems to occur in the plasma compartment as neither statin altered GPI-PLD mRNA levels in HepG2 cells. Serum glycosylphosphatidylinositol-specific phospholipase D levels are regulated by statins and may represent an additional biochemical mechanism for affecting serum triglyceride levels.

Glycosylphosphatidylinositol-specific phospholipase d in nonalcoholic Fatty liver disease: a preliminary study.

CONTEXT: Recent studies demonstrated that de novo lipogenesis is increased in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD also have plasma lipid abnormalities. These lipid abnormalities may in part be related to insulin resistance, which is common in patients with NAFLD. Insulin resistance is associated with alterations in proteins involved in lipid metabolism including glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), which is involved in triglyceride metabolism. OBJECTIVE: The objective of the study was to determine whether alterations in serum and hepatic levels of GPI-PLD occur in patients with NAFLD. DESIGN AND PATIENTS: We examined the following: 1) levels of serum GPI-PLD in nondiabetics with nonalcoholic steatohepatitis, compared with matched controls; 2) hepatic expression of GPI-PLD mRNA in patients with normal liver or NAFLD; and 3) effect of overexpressing GPI-PLD vs. beta-galactosidase (control) on global gene expression in a human hepatoma cell line. RESULTS: The serum levels of GPI-PLD were significantly higher in patients with nonalcoholic steatohepatitis than in matched controls (119 +/- 24 vs.105 +/- 15 microg/ml, P = 0.047). The hepatic expression of GPI-PLD mRNA was increased nearly 3-fold in NAFLD patients, compared with patients with normal liver (3.1 +/- 2.6 vs. 1.1 +/- 1.0 arbitrary units per microgram total RNA, P = 0.026). Finally, overexpressing GPI-PLD was associated with an increase in de novo lipogenesis genes. CONCLUSIONS: Patients with NAFLD have elevated serum levels and hepatic expression of GPI-PLD, and its overexpression in vitro is associated with increased expression of de novo lipogenesis genes. These results suggest that GPI-PLD may play a role in the pathogenesis of NAFLD and/or its metabolic features and warrants further investigation.

Glycosylphosphatidylinositol-specific phospholipase D influences triglyceride-rich lipoprotein metabolism.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a minor HDL-associated protein. Because many minor HDL-associated proteins exchange between different lipoprotein classes during the postprandial state and are also involved in triglyceride (TG) metabolism, we hypothesized that GPI-PLD may play a role in the metabolism of TG-rich lipoproteins. To test this hypothesis, we examined the distribution of GPI-PLD among lipoprotein classes during a fat tolerance test in C57BL/6 and LDL receptor-deficient (LDLR(-/-)) mice fed either a chow or high-fructose diet. In the fasting state in wild-type mice fed a chow diet, GPI-PLD was only present in HDL, whereas in LDLR(-/-) mice GPI-PLD was present in HDL and intermediate-density lipoproteins (IDL)/LDL. During the fat tolerance test, there was no change in total serum GPI-PLD levels in either model; however, a significant amount of GPI-PLD appeared in both VLDL (0.5-1% of total GPI-PLD) and IDL/LDL (5-10% of total GPI-PLD) in both models. The high-fructose diet increased both fasting and postprandial TG and serum GPI-PLD levels in both strains as well as the amount of GPI-PLD in VLDL. To determine whether GPI-PLD plays a direct role in TG metabolism, we increased liver GPI-PLD expression in C57BL/6 mice by adenovirus-mediated gene transfer, which resulted in a sevenfold increase in serum GPI-PLD levels. This change was associated with an increase in fasting (30%) and postprandial TG (50%) and a twofold reduction in TG-rich lipoprotein catabolism compared with saline or control adenovirus-treated mice. These studies demonstrate that GPI-PLD affects serum TG levels by altering catabolism of TG-rich lipoproteins.

[The plasma levels of orexigenic peptides and human platelets phospholipase D activity in anorexia nervosa patients]. / Stezenie wybranych peptydów oreksygenicznych w osoczu a aktywnosc fosfolipazy d w plytkach krwi u chorych z jadlowstretem psychicznym.

UNLABELLED: Ghrelin, orexins A and B (OXA, OXB) are neuropeptides engaged in the regulation of energy balance stimulating appetite and feeding. Disturbances in their activity are proposed to be involved in pathomechanism of eating disorders, particularly in anorexia nervosa (AN). The intracellular mechanism of the peptides action remains unclear. It is considered whether the orexigenic peptides may act through second messengers related to phospholipase D (PLD). The aim of the study was to find a hypothetical relation between ghrelin, OXA and OXB levels and human platelets PLD activity. MATERIAL AND METHODS: 25 AN females and 20 healthy controls (mean age 17.6 and 18.6, mean BMI 15.03 and 21.41 kg/m(2), respectively) were examined. OXA and OXB plasma levels and total ghrelin plasma level was determinated with RIA. PLD activity in homogenized blood platelets was assessed with modified fluorometric method. All values are presented as the mean values. The data were analyzed using Student-t test, non-parametric U-Mann-Whitney test and Spearman correlation. The p < 0.05 was accepted as the level of significance. RESULTS: There is a correlation between analyzed neuropeptides and PLD in AN patients. CONCLUSION: In AN patients it is not possible to exclude influence orexins and ghrelin on platelet PLD activity.

[Effect of GPI-PLD on adhesion function of bone marrow mononuclear cell from patients with myeloid leukemia and its mechanism].

To explore the effect of glycosyl-phosphatidyl inositol-specific phospholipase D (GPI-PLD) on the adhesion function of bone marrow mononuclear cell from patients with myeloid leukemia and analyze its mechanism, the activity of GPI-PLD in bone marrow mononuclear cell from the patients were measured by using GPI-anchored placental alkaline phosphatase (PLAP) as substrate and Triton-X114 partitioning; the adhesion rate and CD24 expression of these cells were measured by MTT and immunohistochemical method respectively, when these cells were or were not treated by 1 mmol/L 1,10-phenanthroline for 5 hours. The results showed that the GPI-PLD activity of bone marrow mononuclear cells from the patients was significantly inhibited after being treated by 1 mmol/L 1, 10-phenanthroline for 5 hours [(42.08 +/- 7.21)% vs (5.4 +/- 2.96)%], while the adhesion rate and the expression of CD24 of these cells were increased [(49.78 +/- 26.73)% vs (61.19 +/- 29.14)%, (16.02 +/- 9.68)% vs (18.5 +/- 11.14)%, respectively)]. It is concluded that depression of GPI-PLD activity can increase the adhesion rate of bone marrow mononuclear cells from the patients while the CD24 expression is enhanced.

Lysophosphatidic acid increases phosphatidic acid formation, phospholipase D activity and degranulation by human neutrophils.

I-oleoyl-sn-glycero-3-phosphate, a lysophosphatidic acid (LPA), in serum is a biologically active lipid and has multiple functions depending on the cell types. Several studies have shown that LPA stimulates phospholipase D (PLD) activity in fibroblasts and prostate cancer cells in culture. PLD plays a central role in regulating neutrophil functions. One of the functions of the lipid product, phosphatidic acid (PA), of PLD action in neutrophils is to promote degranulation. In the present study, we examined the effect of LPA on PLD activity and degranulation by human neutrophils. The results show that exogenous LPA increased PA formation, PLD activity and degranulation by human neutrophils in a time and concentration dependent manner. These findings suggest that LPA released from activated platelets during blood clotting may participate in bacterial killing and wound healing process. On the other hand, augmented LPA production might be involved in inflammation, causing damage of the host tissues.

Changes in phospholipase D activity of leukocytes during human systemic inflammatory response syndrome induced by cardiopulmonary bypass.

OBJECTIVE: To investigate the fluctuations in arterial leukocyte phospholipase D (PLD) activity during the perioperative period of open heart surgery under cardiopulmonary bypass (CPB), and the relationship between PLD activity and systemic inflammatory response induced by CPB. METHODS: Arterial blood was obtained from 26 patients undergoing open heart surgery at 8 different time points during the perioperative period, from which leukocytes were isolated for determination of PLD activity, CD11b expression and myeloperoxidase (MPO) activity. Plasma IL-6, IL-8 and C-reactive protein were also determined. The 26 cases were retrospectively divided into 3 groups according to perfusion time in order to detect the possible influences of CPB on PLD activity and IL-6 and IL-8 levels. RESULTS: When the ascending aorta was declamped, average arterial leukocyte PLD activity was 0.305 +/- 0.132 nmol choline.min(-1).mg(-1), 5.0 times higher of the pre-CPB value, and remained (5.4 times higher of the pre-CPB level) at 72 hours after CPB. Leukocyte CD11b expression and plasma IL-6 and IL-8 levels increased significantly at the end of CPB, while MPO activity and C-reactive protein concentration reached their peaks at 1 and 24 hours, respectively, after CPB. At the end of CPB, the arterial leukocyte PLD activity of patients whose CPB duration was longer than 90 minutes were 1.82- and 1.74-fold that of the other two groups with CPB lasting between 90 and 60 minutes and less than 60 minutes. CONCLUSIONS: Arterial leukocyte PLD activity rises significantly in CPB and its elevation is earlier and more persistent than other inflammation-related indicators tested; longer CPB duration leads to higher leukocyte PLD activity at the end of CPB. These results imply that PLD could be a new target for prevention of systemic inflammatory response induced by CPB.

Phosphorylation decreases trypsin activation and apolipoprotein al binding to glycosylphosphatidylinositol-specific phospholipase D.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present in plasma as an apolipoprotein and as a cell-associated lipase. GPI-PLD mRNA levels are regulated, but it is unclear if posttranslational mechanisms also regulate GPI-PLD function. We examined the effect of protein kinase A phosphorylation on human serum GPI-PLD activity, trypsin activation, and apolipoprotein AI binding. Protein kinase A phosphorylation did not activate GPI-PLD activity in vitro, nor did phosphorylated GPI-PLD cleave a GPI-anchored protein from intact porcine erythrocytes. Trypsin cleaves the C-terminal beta propeller of purified human serum GPI-PLD to generate three immunodetectable fragments (75, 28, and 18 kDa) in association with a 12-fold increase in enzyme activity. After phosphorylation, the amounts of 28- and 18-kDa fragments were markedly decreased with trypsin treatment, and activity was only increased five-fold. Phosphorylation also inhibits binding of GPI-PLD to apolipoprotein AI. These data are the first demonstrating that phosphorylation may regulate GPI-PLD interaction with other proteins.

Screening and modulation of extracellular signals by mucous barrier. Serum glycosylphosphatidylinositol phospholipase D (GPI-PLD) releases protective mucous barrier from oral mucosa.

BACKGROUND: Performance of mucosal epithelial barrier is modified by numerous agents that exert effects on mucin- Mucin Binding Protein (MBP) complex. The aim ofthe studies described was to determine the nature of the damage or modification of oral mucous barrier by the short-term exposure to ethanol. METHODS: Culture of rat buccal mucosa in the presence of ethanol and [3H]-labeled proline and palmitate revealed substantial decrease in MBP synthesis and the release of MBPto the medium. The radioscanning of the samples prepared from the culture medium and the apical epithelial membranes subjected to SDS-PAGE and western blotting disclosed that the released, water soluble 97kDa MBP glycopeptide was labeled with proline and palmitate. When the experiments were conducted in the presence of 5mM EDTA, the GPI-PLD inhibitor, the majority of radiolabeled MBP remained in the membrane-bound form and was extractable with Triton X- 114. The results on the purified GPI-linked MBP degradation by serum enzyme, by the saliva containing serum transudate, and the suppression of the process by inclusion of GPI-PLD-specific inhibitor support our contention that membrane MBP is released to medium by GPI-PLD-like activity. RESULTS: The release of MBP from apical epithelial surfaces was induced by depletion of mucin and the presence of serum-derived GPI-PLD in the tissue homogenate. Strong likelihood exists that under in situ conditions ethanol-induced transudation of serum to saliva provides the vehicle for the transfer of GPI-PLD activity to salivary contents. Defacement of the oral surfaces from mucous barrier signals prospect of lumenal agent influence on the unprotected epithelial exterior, and allows ingression of microbes and untoward acting substances into the organism.

Effects of methylprednisolone and aprotinin on phospholipase D activity of leukocytes in systemic inflammatory response induced by cardiopulmonary bypass.

AIM: To investigate the role of leukocyte phospholipase D (PLD) in systemic inflammatory response induced by cardiopulmonary bypass (CPB) and the effects of methylprednisolone and aprotinin on leukocyte PLD activity. METHODS: Forty-two patients who received CPB open heart surgery were divided into 3 groups: methylprednisolone group, aprotinin group, and control group. Arterial blood (10 mL) was collected for assay of leukocyte PLD activity, myeloperoxidase (MPO) activity, and CD11b expression at 8 different time points in perioperative period. Plasma IL-6, IL-8, and C-reactive protein levels were also determined. RESULTS: At the time point of ascending aorta declamped, leukocyte PLD activity for control group was (18 +/- 8) nmol choline . h-1 . mg-1, which was higher than that of pre-CPB (P < 0.01); the PLD activity for methylprednisolone group was (10 +/- 6) nmol choline . h-1 . mg-1 that was lower than control (P < 0.05), while it had no statistical difference compared with that of pre-CPB. In methylprednisolone group, PLD activity elevation was postponed to the time point of CPB stopped. There was no statistical difference in PLD activity between aprotinin group and control (P > 0.05). After administration of methylprednisolone or aprotinin, leukocyte CD11b expression, plasma IL-6, IL-8, C-reactive protein levels, and MPO activity decreased by different extent. CONCLUSION: Leukocyte PLD activity was elevated significantly in systemic inflammatory response induced by CPB and methylprednisolone partially blunted the CPB-induced inflammatory response by inhibiting PLD activity.

Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins.

We have previously demonstrated that platelets in polycythaemia vera (PV) exhibit decreased aggregation after stimulation with platelet activating factor (PAF) and reduced expression of GPIIIa on both resting and stimulated platelets. In the present study, we investigated if these results were related to changes in the mobilization of intracellular calcium, activation of phospholipase D (PLD) or amounts of GPIIIa and the intracellular tyrosine kinases Fak, Syk, Grb2, Shc and rhoA. Intracellular calcium levels were not different in resting platelets from 14 PV patients and 15 healthy controls (median 43 nmol/L, range 10-114, vs. 36 nmol/L, range 10-119). After stimulation with PAF (1 micromol/L) an equal increase was seen (125 nmol/L for PV platelets, range 67-257, vs. 113 nmol/L for controls, range 60-250). Also formation of phosphatidyl ethanol (PEt) was similar after exposure to 0.5 U/ml thrombin (0.28% PEt of total phospholipid, range 0.16-1.10, vs. 0.24 for controls, range 0.11-2.3) and 1 micromol/L PMA (0.25, range 0.16-0.32, vs. 0.14, range 0.09-0.6). In contrast to the reduced amount of GPIIIa on the surface of PV platelets, immunoblotting on whole cell lysates showed no reduction in PV patients compared to controls, indicating the possibility of an impaired incorporation of GPIIIa to the cell membrane. Levels of Fak, Syk, Shc, Grb2 and rhoA appeared equal in patients and controls. Similar intracellular proteins were tyrosine phosphorylated after stimulation with thrombin, PAF and PMA. In summary, defective platelet aggregation after stimulation with PAF is caused by neither defective mobilization of intracellular calcium nor, in contrast to the situation in PV granulocytes, an impaired activation of PLD. Moreover, no apparent differences in the intracellular amounts of Fak, Syk Shc, Grb2 and rhoA could be detected between PV and control platelets.

Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases.

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527)

Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils.

Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPgammaS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.

2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils.

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.