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T M McIntyre

Publications and source records attributed to T M McIntyre.

At least 145 records · Page 8Linked to original sources
PubMed1987

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils.

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.

Amines↗
PubMed1987

Production of platelet-activating factor by stimulated human polymorphonuclear leukocytes. Correlation of synthesis with release, functional events, and leukotriene B4 metabolism.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation that is synthesized by several human cell types including polymorphonuclear leukocytes (PMN). We examined the synthesis and release of PAF by stimulated human PMN under several conditions, assayed by the incorporation of [3H]acetate into PAF and by bioassay. PAF synthesis was induced by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and N-formyl-methionyl-leucyl-phenylalanine (FMLP) with the relative order of potency IoA much greater than OpsZ greater than FMLP. A variety of other agonists, including phorbol myristate acetate, an activator of protein kinase C and of PMN functional responses, did not stimulate PAF synthesis. PAF synthesis by PMN in response to IoA, OpsZ, and FMLP was concentration- and time-dependent but release of the phospholipid was not: little PAF (1 to 10%) was released from PMN in suspension regardless of the total amount produced, the agonist, its concentration, the time of incubation, or the concentration of extracellular albumin. This was also the case with functionally altered neutrophils that had been "primed" with cytochalasin B or lipopolysaccharide or that had adhered to surfaces. PAF synthesis was tightly coupled with leukotriene B4 production by adherent PMN as well as by neutrophils in suspension, supporting the hypothesis that the two lipid autacoids may be derived from a common precursor. However, PAF synthesis could be dissociated from aggregation and surface adhesion, indicating that it is not absolutely required for these responses of activated PMN. The total amount of PAF that accumulated, but not the percentage that was released, was altered in adherent PMN compared to cells in suspension. These experiments demonstrate that PAF production and its subsequent processing by human neutrophils are highly regulated events. PAF synthesis is associated with PMN activation, but it is not a requisite for early adhesive responses of neutrophils. Because little of the PAF produced by stimulated PMN is released from the cells, it appears that PAF has an intracellular role in PMN function and/or that it may have novel intercellular effects that do not require release into the fluid phase.

Calcimycin↗
PubMed1987

Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor.

Platelet-activating factor (PAF) is a bioactive phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) synthesized by a variety of mammalian cell types. PAF induces hypotension, and activates neutrophils and platelets, among other actions. Removal of the acetyl moiety abolishes biological activity, so this reaction may regulate the concentration of PAF and its physiological effects. We have studied the significance of this reaction, which is catalyzed in vitro by an acetylhydrolase present in mammalian plasma, blood cells, and tissues. We have shown that the plasma PAF-acetylhydrolase is responsible for the degradation of PAF in whole human blood and that alternate pathways for PAF degradation in plasma or blood cells are negligible. Human plasma PAF-acetylhydrolase is associated with low and high density lipoproteins (LDL and HDL with apoE). We have confirmed that the activity is a stable component of these particles by density gradient ultracentrifugation, chromatography on heparin-agarose, and immunoprecipitation. The LDL-associated activity accounts for most or all of the PAF degradation that occurs in plasma ex vivo, while the HDL-associated activity contributes little to this process. However, the two activities likely are due to a single protein since the HDL- and LDL-associated PAF-acetylhydrolase activities can transfer from one lipoprotein to the other. These transfer processes are pH-dependent and specific, since they only occur from LDL to a well characterized subclass of HDL (apoE-containing HDL) and vice versa. We discuss the equilibrium between the two particles and the role that this process may have in vivo.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1987

Human plasma platelet-activating factor acetylhydrolase. Purification and properties.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1986

Leukotrienes C4 and D4 stimulate human endothelial cells to synthesize platelet-activating factor and bind neutrophils.

Human endothelial cells in culture produced platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with leukotriene C4 or D4 (LTC4 or LTD4). Other arachidonate metabolites did not induce the synthesis of PAF. Accumulation of PAF was a prolonged response with maximal accumulation after 10-20 min of stimulation. Half of this amount remained after 90 min of stimulation. The PAF synthesized by endothelial cells remained associated with these cells. LTC4 and LTD4 also induced the adherence of human neutrophils (polymorphonuclear leukocytes; PMNs) to the endothelial cell monolayer. Adhesion was an endothelial cell-mediated process because PMNs adhered to monolayers that had been stimulated and washed prior to PMN addition, and neither LTC4 nor LTD4 stimulated PMNs in the absence of endothelial cells. The time course of PMN adhesion paralleled that of PAF accumulation by endothelial cells, and exogenously added PAF induced adherence. PMNs specifically desensitized to PAF showed only 39% of the agonist-stimulated adherence of control PMNs. We conclude that the PAF synthesized and retained by LTC4- or LTD4-stimulated endothelial cells may induce the adherence of previously unstimulated PMNs. This process may be relevant to inflammation, thrombosis, and mechanisms of vascular injury, including atherosclerosis.

Cell Adhesion↗
PubMed1986

Thrombin stimulates neutrophil adherence by an endothelial cell-dependent mechanism: characterization of the response and relationship to platelet-activating factor synthesis.

Thrombin, a serine coagulation protease that is generated at sites of tissue injury and inflammation, stimulates the adherence of PMNs and neutrophils to EC. We found that thrombin enhanced the adhesion of neutrophils to primary monolayers of human umbilical vein EC when assayed by the binding of 111Indium-labeled PMNs to the EC, the recovery of unlabeled PMNs after incubation with thrombin-treated EC, and by phase contrast and scanning electron microscopy (SEM). SEM demonstrated that thrombin caused PMNs to intimately adhere to the EC plasma membrane and under some conditions to become polarized. The thrombin-stimulated adherence was a rapid, time-dependent response with an onset within 1 minute of addition of thrombin, a peak at 5-10 minutes, and a decline thereafter. The response was concentration-dependent over the range 0.01-2 U/ml thrombin, and required active thrombin. Prothrombin, factor Xa, and fibrinogen were not effective. Thrombin-stimulated PMN adherence was dependent on the EC, because thrombin did not significantly stimulate neutrophils to adhere to albumin-coated petri dishes, subendothelial matrices, or primary cultures of smooth muscle cells. Human EC, when treated with thrombin, also produce platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The concentration-response relationships and time courses for thrombin-stimulated PMN adherence and PAF production were tightly correlated. Furthermore, PAF itself stimulated the adherence of PMNs to EC, and pretreatment of PMNs with PAF selectively inhibited their adherence response to thrombin. These findings demonstrate two novel biologic activities of thrombin, the stimulation of EC-dependent adherence of PMNs and the production of PAF by EC, and suggest that they are functionally related. In addition, they suggest that thrombin may act as a plasma-derived humoral mediator of inflammation under some conditions.

Blood Coagulation↗
PubMed1985

Production of platelet-activating factor by human vascular endothelial cells: evidence for a requirement for specific agonists and modulation by prostacyclin.

Primary cultures of confluent endothelial cells derived from human umbilical veins produce platelet-activating factor (PAF) (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with appropriate agonists. Highly purified human thrombin and calcium ionophore A23187 stimulate the incorporation of [3H] acetate into a lipid product that has been identified as PAF by its behavior in thin-layer chromatographic and high-performance liquid chromatographic systems, the presence of characteristic biologic activity, and appropriate response to phospholipases. A number of other humoral mediators, examined because they directly influence the activity of vascular cells or because they may mediate endothelial injury, do not stimulate PAF production by endothelial cells. This indicates that the synthesis of PAF by cultured human endothelial cells is a response to specific agonists and is not an unregulated event that occurs as a result of nonspecific cellular perturbation. The PAF produced by thrombin-treated endothelial cells is a potent stimulus for platelet activation, as assayed by the aggregation of human platelets in autologous plasma. The production of PAF by endothelial monolayers is attenuated by prostacyclin, another product of stimulated endothelial cells. Conversely, PAF production is enhanced by treatment of the endothelial cells with indomethacin, an inhibitor of prostacyclin synthesis from arachidonic acid, indicating that endogenously generated prostacyclin may modulate PAF synthesis. The potential to synthesize PAF, a unique lipid autocoid that stimulates the activation of both platelets and polymorphonuclear leukocytes, suggests that endothelial cells can directly influence the activity of these circulating effector cells. This biologic potential may be important in the interaction of the endothelium with circulating blood cells in physiologic conditions and in syndromes of vascular injury.

Cells, Cultured↗
PubMed1985

Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate.

Cultured human endothelial cells synthesize prostacyclin (PGI2), a potent inhibitor of platelet function, when stimulated with histamine, bradykinin, or ATP. Paradoxically, we report that these agonists also induced the rapid and sustained synthesis of platelet-activating factor (PAF) by endothelial cells. In fact, the synthesis of this potent activator of platelets and neutrophils was induced by stimulation of the same receptor subtype that induced PGI2 synthesis: stimulation of a histamine H1 or a bradykinin B2 receptor induced both PAF and PGI2 synthesis. However, two physiologically important differences exist between the production of PAF and PGI2 by endothelial cells. The synthesis of PGI2 proceeded for only 7.5 min before the abrupt termination of synthesis, whereas the synthesis of PAF was clearly detectable even 45 min after stimulation. Although maximal accumulation of PAF occurred after 10-15 min of stimulation, the prolonged synthesis resulted in the presence of PAF for up to 1 h after stimulation. Secondly, whereas PGI2 was released from the cell monolayer, PAF remained cell-associated without significant release to the external medium. Endothelial cell-generated PAF, therefore, does not function as a hormone. The prolonged association of this potent activator of platelets and neutrophils with endothelial cells may mediate some of the inflammatory properties of histamine and bradykinin. It may also be a factor in the formation of a thrombogenic vascular surface, an event suggested to play a primary role in the pathogenesis of thrombosis and atherosclerosis.

Adenosine Triphosphate↗
PubMed1985

Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.

Highly purified human thrombin stimulates the adherence of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC). When Indium-labeled PMNs were incubated with primary monolayers of cultured human umbilical vein EC, the basal adherence was 10 +/- 1% of the PMNs at 5 min. Addition of thrombin (2 U/ml) increased the mean adherence to 42 +/- 15%. Enhanced neutrophil adherence in response to thrombin was confirmed by experiments with unlabeled leukocytes, examined by phase contrast and scanning electron microscopy. The action of thrombin was on the EC, since it did not directly stimulate PMN adhesiveness when measured by aggregation or by adherence to nylon fiber columns. Furthermore, enhanced neutrophil adherence occurred when endothelial monolayers were treated with thrombin and washed before adding 111Indium (111In)-labeled PMNs. Thrombin that had been inactivated with antithrombin III and heparin did not enhance neutrophil adherence. Prothrombin, Factor Xa, and fibrinogen were also ineffective. The stimulated adherence of PMNs was maximal 5 min after incubation of the EC with thrombin, and decreased thereafter. The response was dose-dependent, with half-maximal stimulation at 0.2-0.25 U thrombin/ml. The enhanced PMN adherence caused by thrombin may result in part from the production of platelet-activating factor (PAF) by the stimulated EC since thrombin-stimulated EC synthesize PAF with a time course and concentration dependence that are similar to the time and concentration relationships for thrombin-stimulated PMN adherence, PAF itself promoted neutrophil adherence to the EC monolayers, and pretreatment of PMNs with PAF decreased the adherence stimulated by thrombin and PAF, but not adherence stimulated by N-formylmethionyl-leucyl-phenylalanine and C5a fragments, which indicates specific desensitization of PAF-mediated adherence. These studies demonstrate the endothelial cell-dependent stimulation of PMN adherence by thrombin, a novel mechanism of enhanced leukocyte adherence that may be important in interactions between the coagulation and inflammatory systems.

Cell Adhesion↗
PubMed1984

Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin.

Cultured human endothelial cells produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) when stimulated with human thrombin. The response to thrombin is dose dependent, with a half-maximal effect at 0.17 unit/ml. The product is identified as PAF by the incorporation of radiolabeled precursors, its behavior in chromatographic systems, the recovery of biological activity, and the effect of treatment with phospholipase A2. Incorporation of [3H]acetate into PAF is maximal by 5 min and decreases thereafter. Endothelial cells produce 0.10-0.17 nmol of PAF per 10(6) cells in a 5-min exposure to thrombin, as judged by the amount of neutrophil-aggregating activity. The production of this potent agonist for platelet activation and neutrophil chemotaxis by endothelial cells suggests that it may play a role in the maintenance of vascular integrity and perhaps in pathological events such as thrombosis and atherogenesis.

Acetates↗
PubMed1982

Somatic cell cloning in polyester stacks.

Single somatic cells, including fibroblasts, myelomas, and hybridomas, proliferate normally when trapped between a plastic dish and a disc of polyester cloth. Contact between the overlay and the plastic for 8-16 days results in identical colony patterns on the cloth and the plate. When several cloth discs are simultaneously stacked over Chinese hamster ovary cells, three or four-high resolution colony copies can be generated from a single master dish. The colonies on the cloth can be analyzed by radiochemical methods [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] or by "replica plating" to a new disc. The use of polyester cloth, singly or in stacks, has several major advantages over previous techniques for somatic cell replica plating, including: (i) broad applicability to diverse cell lines such as fragile membrane mutants of Chinese hamster ovary cells and relatively nonadherent myelomas or hybridomas; (ii) the possibility of generating multiple copies of the same colony population, allowing simultaneous analysis for several enzymes or cellular components; and (iii) superior resolution and transfer efficiency in copying colony patterns from one surface to another. The remarkable capacity of animal cell colonies to proliferate upward through "polyester stacks" may reflect chemotropic movement of individual cells and opens new approaches to somatic cell genetics.

Animals↗
PubMed1978

Escherichia coli mutants defective in membrane phospholipid synthesis: binding and metabolism of 1-oleoylglycerol 3-phosphate by a plsB deep rough mutant.

Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's.

Cell Membrane↗