""""""""Priming"""""""" of granulocytes (PMNL) signifies an increased responsiveness to stimulation in vitro. The respiratory burst is important to the bactericidal activity of PMNL and priming of the burst may be an important aspect of host defense in such patients. Normal PMNL can be primed in vitro by preincubation with certain agonists, followed by stimulation with a second agonist. We hypothesize that priming involves synergistic convergence of separate, collateral stimulus-response coupling mechanisms. Since it is effective for over 10 minutes, priming can be used to dissect transductional mechanisms into sequential events. This facilitates analysis of causal relationships among the many """"""""early events"""""""" occurring after stimulation of membrane receptors. 1,2-diacylglycerols prime both initiation (lag time, initial rate) and duration of the burst stimulated by formyl-met-leu-phe or by phagocytosis. 1-0-alkyl-2-acylglycerols prime only initiation. Thus, initial activation of the NADPH oxidase and the maintenance of ongoing oxidase activation are separable and will be probed with this model system. Priming and primed stimulation are hypothesized to involve interactions between 1) diglycerides, 2) arachidonic acid (AA) release and metabolism, 3) altered membrane structure, 4) synergy between DGs and/or AA with Ca++ in activation of Ca++ -dependent enzymes. These synergistic mechanisms are proposed to cause 5) structural modification (e.g., protein phosphorylation) and 6) translocation to the plasma membrane of intracellular components with assembly and activation of the NADPH oxidase. The project is therefore divided into 6 specific aims as follows: 1) Using synthetic diglyceride analogs, define the molecular specificity for priming of initiation and prolongation of the burst. Use these models in aims 2-6. 2) Determine the role of PLA2 activation by direct measurement of AA release and metabolism; determine that primed-stimulation also involves increased sensitivity to AA-mediated events. 3) Determine physical membrane changes induced by priming and primed stimulation using several fluorescent and resonance methods. 4) Determine changes in cytosolic (Ca++), 45Ca fluxes, and the effects of chelation of intracellular Ca on each of the parameters. 5) Define protein phosphorylation occurring with priming and primed stimulation. 6) Define translocation of oxidase components, oxidase kinetics and changes in concentration of the oxidase substrate, NADPH, in each model system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI014929-09
Application #
3125928
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1979-09-01
Project End
1992-11-20
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
9
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
Bauldry, S A; Nasrallah, V N; Bass, D A (1992) Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ. J Biol Chem 267:323-30
Bauldry, S A; Elsey, K L; Bass, D A (1992) Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. J Biol Chem 267:25141-52
Agwu, D E; McPhail, L C; Sozzani, S et al. (1991) Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase. J Clin Invest 88:531-9
Bauldry, S A; Wykle, R L; Bass, D A (1991) Differential actions of diacyl- and alkylacylglycerols in priming phospholipase A2, 5-lipoxygenase and acetyltransferase activation in human neutrophils. Biochim Biophys Acta 1084:178-84
Bauldry, S A; Bass, D A; Cousart, S L et al. (1991) Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation. J Biol Chem 266:4173-9
Nieto, M L; Venable, M E; Bauldry, S A et al. (1991) Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction. J Biol Chem 266:18699-706
Bauldry, S A; McCall, C E; Cousart, S L et al. (1991) Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming. J Immunol 146:1277-85
Wheeler, J G; Winkler, L S; Seeds, M et al. (1990) Influenza A virus alters structural and biochemical functions of the neutrophil cytoskeleton. J Leukoc Biol 47:332-43
Bauldry, S A; Wykle, R L; Bass, D A (1988) Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe. J Biol Chem 263:16787-95
Bass, D A; McPhail, L C; Schmitt, J D et al. (1988) Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C. J Biol Chem 263:19610-7

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