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S M Prescott

Publications and source records attributed to S M Prescott.

At least 109 records · Page 6Linked to original sources
PubMed1994

Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes.

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation and atherosclerosis. A specific degradative enzyme found in plasma, PAF acetylhydrolase, plays important roles in various pathophysiological events induced by PAF. Human macrophages and Hep G2 cells secrete PAF acetylhydrolase with characteristics identical to the plasma activity. Other investigators reported that apolipoprotein B may possess phospholipase A2 activity, which suggested that apolipoprotein B might be a zymogen for PAF acetylhydrolase. However, while macrophages express PAF acetylhydrolase activity, we did not detect cDNAs for apolipoprotein B in a cDNA library from these cells, indicating that macrophages do not express this protein. In contrast, Hep G2 cells had high levels of cDNA for apolipoprotein B, as expected. We next injected Xenopus laevis oocytes with poly(A)+ RNA extracted from cultured human macrophages and Hep G2 cells. Twenty-five to 50% of Xenopus oocytes injected with poly(A)+ RNA from macrophages or Hep G2 cells secreted a PAF acetylhydrolase activity (1.0-7.8 nmol/ml per h) that also utilized a synthetic oxidized phospholipid as substrate. The activity secreted by poly(A)+ RNA-injected oocytes associated with lipoproteins and transferred between the particles in a pH-dependent manner, much like the plasma activity. These experiments establish that the properties of the enzyme released from poly(A)+ RNA-injected oocytes are identical to those of the plasma form of PAF acetylhydrolase and that the activity detected is not the expression of a domain in apolipoprotein B.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1994

CD11b/CD18 integrin and a beta-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes.

We determined the mechanism by which opsonized zymosan particles, which are derived from yeast and composed of carbohydrate polymers, stimulate platelet-activating factor (PAF) synthesis by monocytes. A role for CD11b/CD18 was demonstrated because antibodies to this integrin decreased PAF synthesis, zymosan bearing only a ligand for CD11b/CD18 (iC3b) induced the synthesis of PAF, and monocytes that did not express CD11b/CD18 produced much less PAF than control monocytes. Ligation of CD11b/CD18 was not sufficient for PAF synthesis suggesting that an additional receptor was involved. Monocytes are known to bind beta-glucan which is a major component of zymosan. Opsonized beta-glucan particles stimulated the synthesis of PAF, and a soluble form of beta-glucan partially inhibited PAF synthesis in response to opsonized zymosan. Two lines of evidence suggested that the beta-glucan receptor mediating this response was distinct from CD11b/CD18. First, CD11b/CD18-deficient monocytes produced PAF when stimulated by zymosan opsonized with isolated C3b, a molecule that binds to complement receptor type 1 (CD35). Second, inducing contact of monocytes with zymosan by centrifugation resulted in PAF synthesis that was not inhibited by antibodies to CD11b/CD18. The combination of soluble beta-glucan and antibodies to CD11b/CD18 completely blocked PAF synthesis in response to opsonized zymosan. Together, these results demonstrate that induction of maximal PAF synthesis by serum-opsonized zymosan requires the concerted interactions of monocyte receptors for iC3b and beta-glucan. Additionally, they suggest that CD11b/CD18 facilitates binding of the particle and that a beta-glucan receptor transduces the activation signal.

Antigens, CD↗
PubMed1994

The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation. Differential induction of GM-CSF, IL-8, and E-selectin expression.

Brown recluse spider (Loxosceles reclusa) venom induces severe dermonecrotic lesions. The mechanism for this is unknown but presents an interesting paradox: necrosis is completely dependent on the victim's neutrophils, yet neutrophils are not activated by the venom. We show Loxosceles venom is a potent, but disjointed, endothelial cell agonist. It weakly induced E-selectin expression, but not intercellular adhesion molecule-1 or IL-6 expression, yet significantly stimulated release of IL-8 and large amounts of GM-CSF by 4 h. In contrast, TNF strongly induced all of these, except for GM-CSF. PMN bound to E-selectin on venom-activated endothelial cells, apparently via counterreceptors different from those that bind E-selectin on TNF alpha-activated monolayers. Notably, PMN bound venom-activated monolayers only at intercellular junctions, did not polarize, and completely failed to migrate beneath the monolayer. Despite this, bound PMN demonstrated increased intracellular Ca2+ levels and secreted primary and secondary granule markers. The latter event was suppressed by sulfones used to treat envenomation. We have defined a new endothelial cell agonist, Loxosceles venom, that differentially stimulates the inflammatory response of endothelial cells. This, in turn, leads to a dysregulated PMN response where adhesion and degranulation are completely dissociated from shape change and transmigration.

Animals↗
PubMed1994

Proliferation-dependent changes in release of arachidonic acid from endothelial cells.

Stimulation of endothelial cells resulted in release of arachidonic acid from phospholipids. The magnitude of this response decreased as the cells became confluent and the change coincided with a decrease in the percentage of cells in growth phases (G2+M); this was not a consequence of time in culture or a factor in the growth medium. Preconfluent cells released approximately 30% of arachidonic acid; confluent cells released only 6%. The decreasing release of arachidonic acid was demonstrated using metabolic labeling, mass measurements of arachidonic acid, and measurement of PGI2. The decrease was not due to a changing pool of arachidonic acid, and mass measurements showed no depletion of arachidonic acid. Release from each phospholipid and from each phospholipid class decreased with confluence. Conversion of confluent cells to the proliferative phenotype by mechanical wounding of the monolayer caused increased release of arachidonic acid. Potential mechanisms for these changes were investigated using assays of phospholipase activity. Phospholipase A2 activity changed in concert with the alteration in release, a consequence of changes in phosphorylation of the enzyme. The increased release of arachidonic acid from preconfluent, actively dividing cells may have important physiologic implications and may help elucidate mechanisms regulating release of arachidonic acid.

Animals↗
PubMed1993

Growth-dependent changes in arachidonic acid release from endothelial cells are mediated by protein kinase C and changes in diacylglycerol.

When stimulated, endothelial cells release arachidonic acid from phospholipids and oxidize it to eicosanoids. The rate-limiting step in this pathway is the initial release step, catalyzed by phospholipase(s), a process that exhibits growth-dependent changes. We examined the role of protein kinase C (PKC) as a regulator of this process. Activators and inhibitors of protein kinase C, used at different growth states, demonstrated distinct differences in their effects on arachidonic acid release, consistent with a growth-dependent change in PKC activity (with greater activity in proliferating cells compared with quiescent cells). Although immunoreactive PKC was slightly greater in the proliferating cells, there was a more striking redistribution of PKC activity between cytosol and membrane. To identify the cause, we measured the diacylglycerol (DG) content and found that DG concentrations decreased as cells progressed from preconfluence to confluence. Further studies demonstrated increases in DG kinase and DG lipase in confluent compared with preconfluent cells, consistent with the alterations in DG content. These findings suggest that growth-dependent changes in DG lipase and DG kinase activities regulate basal DG levels and PKC activity. The consequent alteration in PKC activity regulates the growth-dependent changes in arachidonic acid release.

Animals↗
PubMed1993

P-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage.

We report that the adhesion receptor P-selectin can be metabolically labeled with [3H]palmitic acid in human platelets. Analysis of alkaline methanolysis products from labeled protein demonstrated that the radioactivity associated with P-selectin was covalently bound palmitic acid. [3H]Palmitic acid was cleaved by hydroxylamine treatment at neutral pH and by reducing agents, indicating that acylation occurred through a thioester linkage. Both stearic acid and palmitic acid were detected by gas chromatography-mass spectrometry analysis of alkaline hydrolysates of purified P-selectin. Deletion or mutation of Cys766 eliminated [3H] palmitic acid labeling of P-selectin in transfected COS-7 cells. We conclude that the cytoplasmic domain of P-selectin is acylated at Cys766 through a thioester bond. Fatty acid acylation may regulate intracellular trafficking or other functions of P-selectin.

Acylation↗
PubMed1993

Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines.

Prostaglandin endoperoxide synthase (PHS) catalyzes the committed step in the biosynthesis of prostaglandins and thromboxane. We recently observed dissociation of PHS activity and enzyme mass measured in an immunoassay of endothelial cells exposed to tumor necrosis factor. These data and observations by others suggested that endothelial cells express an alternate PHS. We now report the molecular cloning of human PHS type II from an endothelial cell cDNA library. The protein encoded by this cDNA shares 61% identity with the human PHS I. Southern analysis demonstrated a single copy of PHS II and we found a polymorphism in approximately 5% of the population. PHS II mapped to chromosome 1, in contrast to PHS I, which is on chromosome 9. The PHS II cDNA hybridized strongly to a 4.3-kilobase (kb) message from endothelial cells. Stimulation of the cells with tumor necrosis factor, phorbol 12-myristate 13-acetate, lipopolysaccharide, or interleukin-1 increased mRNA levels for PHS II, and this change correlated well with increased prostacyclin biosynthesis. Cycloheximide induced PHS II mRNA without a corresponding activity increase demonstrating that translation of the 4.3-kb message is required for increased prostacyclin biosynthesis. We conclude that expression of PHS II may have important pathophysiological effects in the vasculature.

Amino Acid Sequence↗
PubMed1993

The platelet-activating factor acetylhydrolase from human erythrocytes. Purification and properties.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is a biologically active phospholipid. Tissues, blood cells, and plasma contain PAF acetylhydrolases (calcium independent phospholipase A2 activities) that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups. They inactivate PAF and thereby determine PAF accumulation. We purified the PAF acetylhydrolase from human erythrocytes 15,600-fold. The enzyme has a molecular weight of 25,000, it behaves as a dimer during gel filtration, and it is a previously uncharacterized cytosolic esterase, as it has a unique amino-terminal sequence. The erythrocyte PAF acetylhydrolase requires the addition of sulfhydryl agents for maximal activity, is inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), NaF, diisopropyl fluorophosphate, diethylpyrocarbonate, p-bromophenacylbromide, and a number of proteases. Antibodies against the purified protein precipitate all PAF hydrolase activity from erythrocyte lysates. The erythrocyte PAF acetylhydrolase is specific for short or oxidized sn-2 acyl residues. It exhibits surface dilution kinetics, suggesting that hydrolysis occurs at lipid interfaces. This suggests that this enzyme acts in vivo as a scavenger of oxidatively fragmented phospholipids that are toxic to the cell.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1993

Juxtacrine intercellular signaling: another way to do it.

Intercellular interactions in which one cell sends a signal to another cell, inducing a change in function of the second cell, are common in morphogenesis, development, inflammation, and repair of the lung and other organs. In juxtacrine intercellular signaling, the molecule that induces the functional changes in the target cell remains associated with the plasma membrane of the signaling cell, rather than acting in the fluid phase. This feature distinguishes juxtacrine signaling from endocrine and paracrine stimulation and provides a mechanism for strict spatial control of activation of one cell by another. Juxtacrine signaling is likely to be common in physiologic events that require tight regulation, and disruption of juxtacrine signaling may lead to pathologic outcomes. In this minireview, general principles as well as several specific examples of juxtacrine signaling are discussed.

Animals↗
PubMed1993

Inflammatory roles of P-selectin.

Polymorphonuclear leukocytes (PMNs) bind rapidly and reversibly to endothelial cells induced to express P-selectin, a glycoprotein that mediates adhesive intercellular interactions. In addition, PMNs adherent to endothelium expressing P-selectin demonstrate an intracellular Ca2+ transient, functionally up-regulate beta-2-integrins (CD11/CD18 glycoproteins), become polarized in shape, and are primed for enhanced degranulation when subsequently stimulated with chemotactic factors. However, P-selectin induces none of these responses directly when used alone, when incorporated into model membranes, or when expressed by transfected cells. The absence of direct activation of the PMNs is not due to competing antiinflammatory effects of P-selectin; instead, purified P-selectin and P-selectin in membranes support agonist-stimulated PMN responses. Furthermore, tethering of PMNs to endothelial surfaces by P-selectin is required for priming to occur efficiently, as shown by experiments with blocking monoclonal antibodies. The priming event is directly mediated by the signaling molecule, platelet-activating factor (PAF), and is inhibited by blocking the PAF receptor on PMNs. Thus, P-selectin and PAF are components of an adhesion and activation cascade, but have distinct roles: P-selectin tethers and captures the PMN, whereas PAF mediates juxtacrine activation. In vivo, selectins may facilitate interaction of target cells with membrane-bound molecules that send intercellular signals, in addition to mediating rolling of leukocytes and other adhesive functions.

Antigens, CD↗
PubMed1992

Activation of the acetyl-coenzyme A:lysoplatelet-activating factor acetyltransferase regulates platelet-activating factor synthesis in human endothelial cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a phospholipid with many physiological actions. It is synthesized by endothelial cells and a variety of others in response to stimulation with receptor-mediated agonists. In endothelial cells it remains associated with the surface of the cell and serves as a signal for adhesive interactions with leukocytes. Thus, its synthesis must be precisely regulated. In previous work we have shown that PAF synthesis is regulated at the initiating step, a phospholipase A2. Here we demonstrate that the subsequent step of PAF synthesis, the acetyl-CoA:lyso-PAF acetyltransferase, is rapidly activated when cells are exposed to thrombin or other agonists. We found that the activity increased from basal values (5 nmol/mg/min) to approximately 3-fold higher within 1 min following the addition of agonists. The enzyme activity returned to basal levels within 10 min. The pattern of activation and inactivation suggested covalent modification of the enzyme. This was supported in experiments in which we showed that homogenates had stable enhanced activity and that there was no evidence for an activator or inhibitor. Pretreatment of the cells with vanadate, an inhibitor of protein phosphatases, markedly prolonged the activation state. In subsequent studies we pretreated intact cells with vanadate to block inactivation of the enzyme and then measured the accumulation of PAF in response to thrombin. We found that it was markedly augmented and prolonged. From this we conclude that the synthesis of PAF in intact cells is regulated by the activity of the acetyltransferase. We characterized requirements for activation of acetyltransferase and found that it was not dependent on the influx of intracellular calcium but that calcium entry did influence the length of time for which the enzyme was activated. The acetyltransferase in endothelial cells was shown to be a specific enzyme that did not catalyze the transfer of long chain acyl groups from acyl-CoA to lysophospholipids and demonstrated modest specificity for the acceptor lysophospholipids. These results suggest that activation of the acetyltransferase is a crucial determinant of the amount of PAF synthesized in activated endothelial cells.

Acetyltransferases↗
PubMed1992

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.

Cell Adhesion↗
PubMed1992

Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells.

The specific precursor for platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, constitutes 10 per cent of the 1-radyl-2-acyl-sn-glycero-3-phosphocholines in endothelial cells. Stimulation of endothelial cells results in accumulation of PAF and its sn-1-acyl- analog (acylPAF), with acylPAF the predominant product. Mass spectrometry confirmed these relative amounts and confirmed that stimulated endothelial cells accumulate 1-3 ng PAF per million cells. These data suggest that stimulated endothelial cells accumulate both PAF and acylPAF and that the PAF synthetic pathway in endothelial cells is not highly selective for the specific PAF precursor (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine).

Animals↗
PubMed1992

Endothelial cell interactions with granulocytes: tethering and signaling molecules.

The adhesion of granulocytes to endothelial cells requires regulated expression of molecules on both the endothelial cell and the granulocyte. These pro-adhesive molecules have diverse structures and mechanisms of expression, and act either to tether the two cells together or as signals that induce activation-dependent adhesion events. Combinations of tethering and signaling molecules regulate endothelial-cell-granulocyte interactions at the endothelial surface.

Cell Adhesion Molecules↗