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Endotoxin-mediated synthesis of nitric oxide is dependent on Gq protein signal transduction.
- Source :
-
Surgery [Surgery] 1997 Aug; Vol. 122 (2), pp. 394-402; discussion 402-3. - Publication Year :
- 1997
-
Abstract
- Background: Nitric oxide (NO) is a ubiquitous multifunctional free radical produced during sepsis, shock, reperfusion injury, and allograft rejection. Many studies are presently evaluating the functional roles of NO production in these settings. However, the signal transduction mechanisms underlying initiation of NO production are largely unknown. This study defines the cell surface receptor proteins that mediate endotoxin-induced NO synthesis in ANA-1 murine macrophages.<br />Methods: Endotoxin (LPS, 10 micrograms/ml) was added to ANA-1 macrophages to induce NO synthesis. In selected instances guanosine 5'-O-(2-thiodiphosphate)-trilithium salt (GOTP), pertussis toxin, cholera toxin, or suramin were added as inhibitors of specific subclasses of heterotrimeric G proteins. Calphostin was added as a protein kinase C inhibitor, and ET-OCH3 was added as a phospholipase C-beta inhibitor. NO release was quantified by measurement of the NO metabolite, nitrite. Membrane guanosine triphosphatase (GTPase) activity was also analyzed. Steady-state levels of inducible nitric oxide synthase (iNOS) mRNA were determined by using reverse transcription-polymerase chain reaction analysis.<br />Results: Inhibition of G protein function by suramin or GOTP significantly decreased synthesis of NO and expression of iNOS mRNA. Pertussis and cholera toxin did not alter NO synthesis, suggesting that the Gi and Gs classes are not involved. Inhibition of protein kinase C or upstream phospholipase C-beta activity decreased NO synthesis, implicating the Gq class of heterotrimeric G proteins.<br />Conclusions: In ANA-1 macrophages, endotoxin-mediated NO synthesis is dependent on heterotrimeric Gq protein-phospholipase C-beta-protein kinase C signal transduction.
- Subjects :
- Animals
Cell Line
Cholera Toxin pharmacology
Enzyme Inhibitors pharmacology
Escherichia coli
Genistein
Guanosine Diphosphate analogs & derivatives
Guanosine Diphosphate pharmacology
Isoflavones pharmacology
Kinetics
Macrophages
Mice
Naphthalenes pharmacology
Pertussis Toxin
Phospholipid Ethers pharmacology
Polymerase Chain Reaction
Protein Kinase C antagonists & inhibitors
Protein-Tyrosine Kinases antagonists & inhibitors
RNA, Messenger biosynthesis
Signal Transduction drug effects
Suramin pharmacology
Tetradecanoylphorbol Acetate pharmacology
Thionucleotides pharmacology
Transcription, Genetic drug effects
Type C Phospholipases antagonists & inhibitors
Virulence Factors, Bordetella pharmacology
Endotoxins pharmacology
GTP-Binding Proteins metabolism
Lipopolysaccharides pharmacology
Nitric Oxide biosynthesis
Nitric Oxide Synthase biosynthesis
Signal Transduction physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0039-6060
- Volume :
- 122
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Surgery
- Publication Type :
- Academic Journal
- Accession number :
- 9288146
- Full Text :
- https://doi.org/10.1016/s0039-6060(97)90032-5