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PMID: 1907286 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Effects of protein I of Neisseria gonorrhoeae on neutrophil activation: generation of diacylglycerol from phosphatidylcholine via a specific phospholipase C is associated with exocytosis.

The Journal of cell biology ·Vol. 114 ·No. 3 ·1991-08-00 ·Pages 433-42

Haines KA, Reibman J, Tang XY, Blake M, Weissmann G

Abstract

Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)

MeSH Terms
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives,pharmacology Adult Bacterial Outer Membrane Proteins/immunology Cells, Cultured Diglycerides/metabolism Enzyme Activation Ethanol/pharmacology Exocytosis Humans Kinetics Lymphocyte Activation N-Formylmethionine Leucyl-Phenylalanine Neisseria gonorrhoeae/immunology Neutrophils/immunology,metabolism Phosphatidylcholines/metabolism Phospholipids/metabolism Porins Propranolol/pharmacology Protein Kinase C/metabolism Type C Phospholipases/metabolism
Chemicals
Bacterial Outer Membrane Proteins Diglycerides Phosphatidylcholines Phospholipids Porins porin protein, Neisseria 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid Ethanol N-Formylmethionine Leucyl-Phenylalanine Propranolol Protein Kinase C Type C Phospholipases 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Haines K A
Department of Medicine, New York University Medical Center, New York 10016.
Reibman J
Tang X Y
Blake M
Weissmann G
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.114.3.433
Published
1991-08-00
Pages
433-42
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2289089
Subset
IM
Grants
NHLBI NIH HHS · 2-RO1HL19721-14 · United States
NIAMS NIH HHS · 5-R37AR11949-23 · United States
External Links
PubMed source DOI: 10.1083/jcb.114.3.433 PMC Full Text View journal details
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