# REGULATION OF GRANULOCYTE SECRETION

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 1993 · $252,586

## Abstract

Our studies will test the hypothesis that during receptor-mediated
activation of adherent or adherent phagocytosing polymorphonuclear
leukocytes (PMN), the NADPH oxidase can be assembled on specific granule
(SG) membranes which contain lactoferrin (LF) and cytochrome b558.  Our
studies will test whether phosphatidic acid (PA) initiates the assembly
of the NADPH oxidase onto SG membranes either prior to or during
exocytosis.  Our studies will be directed to assess the role of
calcium-binding proteins and diacylglycerol (DAG) in initiating and/or
sustaining exocytosis.  We will focus on the metabolic pathways involved
in the assembly of the multicomponent NADPH oxidase onto SG, the
biochemical documentation of NADPH activity on SG, and the subcellular
localization of phosphatidic acid phosphohydrolase.  We will employ
radioactive substrates, which will allow documentation of phospholipase C
or phospholipase D activities, as well as measure mass production of PA
and DAG.  We will monitor assembly of the NADPH oxidase P47phox onto SG
biochemically, immunologically and by employing transmission immune
electron microscopy.  We will document the interrelationship between
release of SG bearing LF and the respiratory burst in adherent PMN
undergoing phagocytosis.  Immunoelectron microscopy will be used to
morphologically assess the assembly of the NADPH oxidase by monitoring
the association of the oxidase component p47Phox with LF bearing SG. We
will employ immunofluorescence intensified-enhanced microscopy to
document the entry of oxidants into phagosomes.  To link delivery of LF
into phagosomes with the respiratory burst we will test the ability of
BAPTA or MAPTAM/ionomycin treatment to affect LF delivery into the
phagosomes by employing resonance energy transfer.  Finally, we will
study the regulation of exocytosis of SG by focusing on cell-free systems
which will allow us to evaluate the role of calcium binding proteins
along with diradylglycerol as cofactors in mediating fusion of SG with
the plasma membrane.  We will purify novel fusogenic proteins by
chromatographic techniques and assess their ability to promote fusion of
SG with PMN plasma membranes using lipid mixing assays.  To confirm
delivery of SG contents into PMN membranes, we will employ immune
electron microscopy.  These studies will document the intracellular
signalling pathways leading to the assembly of the NADPH oxidase on SG
membranes.  They will provide further biochemical understanding of the
factors regulating exocytosis of SG.  The knowledge of both oxidative
metabolism and degranulation in PMN should lead to novel therapeutic
approaches to chronic inflammation.

## Key facts

- **NIH application ID:** 3129571
- **Project number:** 2R01AI020065-11A1
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **SAM.gov UEI:** GNJ7BBP73WE9
- **Principal Investigator:** Laurence Alan Boxer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $252,586
- **Award type:** 2
- **Project period:** 1982-08-01 → 1998-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3129571

## Citation

> US National Institutes of Health, RePORTER application 3129571, REGULATION OF GRANULOCYTE SECRETION (2R01AI020065-11A1). Retrieved via MORVS 2026-10-24 from https://api.morvs.ai/grant/nih/3129571. Licensed CC0.

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